Wnt compositions and methods for purification

ABSTRACT

Disclosed herein are methods, processes, compositions, and kits for generating bone graft materials for use at a site of bone defect that utilizes a composition which contains liposomal Wnt polypeptide, such as liposomal Wnt3a polypeptide, liposomal Wnt5a polypeptide, or liposomal Wnt10b polypeptide. Also disclosed herein are methods, processes, compositions, and kits for enhancing mammalian bone marrow cells that utilizes a composition which contains liposomal Wnt polypeptide, such as liposomal Wnt3a polypeptide, liposomal Wnt5a polypeptide, or liposomal Wnt10b polypeptide.

CROSS REFERENCE

This application claims benefit and is a Continuation of applicationSer. No. 14/910,616 filed Feb. 5, 2016, which is a 371 application andclaims the benefit of PCT Application No. PCT/US2014/058833, filed Oct.2, 2014, which claims benefit of U.S. Provisional Patent Application No.61/885,827, filed Oct. 2, 2013, which applications are incorporatedherein by reference in their entirety.

BACKGROUND OF THE INVENTION

Wnt proteins form a family of highly conserved secreted signalingmolecules that bind to cell surface receptors encoded by the Frizzledand low-density lipoprotein receptor related proteins (LRPs). The WNTgene family consists of structurally related genes which encode secretedsignaling proteins. These proteins have been implicated in oncogenesisand in several developmental processes, including regulation of cellfate and patterning during embryogenesis. Once bound, the ligandsinitiate a cascade of intracellular events that eventually lead to thetranscription of target genes through the nuclear activity of β-cateninand the DNA binding protein TCF (Clevers H, 2004 Wnt signaling:Ig-norrin the dogma. Curr Biol 14: R436-R437; Nelson & Nusse 2004Convergence of Wnt, beta-catenin, and cadherin pathways. Science 303:1483-1487; Gordon & Nusse 2006 Wnt signaling: Multiple pathways,multiple receptors, and multiple transcription factors. J Biol Chern281: 22429-22433).

Wnt proteins are also involved in a wide variety of cellular decisionsassociated with the program of osteogenesis. For example, Wnt proteinsregulate the expression level of sox9, which influences the commitmentof mesenchymal progenitor cells to a skeletogenic fate. Wnt proteinsinfluence the differentiation of cells, into either osteoblasts orchondrocytes. In adult animals, there is evidence that Wnt signalingregulates bone mass. For example, mutations in the human Wnt co-receptorLRPS are associated with several high bone mass syndromes, includingosteoporosis type I, and endosteal hyperostosis or autosomal dominantosteosclerosis, as well as a low bone mass disease,osteoporosis-pseudoglioma. Increased production of the Wnt inhibitorDkkl is associated with multiple myeloma, a disease that has increasedbone resorption as one of its distinguishing features. For furtherdetails, see, S. Minear et al., Wnt proteins promote bone regeneration.Sci. Transl. Med. 2, 29ra30 (2010); and Zhao et al., Controlling the invivo activity of Wnt liposomes, Methods Enzymol 465: 331-47 (2009).

SUMMARY OF THE INVENTION

Disclosed herein, in certain embodiments, is a composition comprising afunctionally active Wnt3a polypeptide and an aqueous solution comprisingliposomes, wherein the functionally active Wnt3a polypeptide comprises alipid modification at an amino acid position corresponding to amino acidresidue 209 as set forth in SEQ ID NO:1. In some embodiments, thefunctionally active Wnt3a polypeptide is not lipid modified at an aminoacid position corresponding to amino acid residue 77 as set forth in SEQID NO:1. In some embodiments, the functionally active Wnt3a polypeptideis integrated into the liposomal membrane. In some embodiments, thefunctionally active Wnt3a polypeptide protrudes from the liposomalmembrane onto the outer surface of the lipid membrane. In someembodiments, the functionally active Wnt3a polypeptide is notincorporated into the aqueous core of the liposome. In some embodiments,the functionally active Wnt3a polypeptide comprises at least about 30%,about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, orabout 95% sequence identity to the amino acid sequence as set forth inSEQ ID NO:1. In some embodiments, the functionally active Wnt3apolypeptide comprises the amino acid sequence as set forth in SEQ IDNO:1. In some embodiments, the concentration of the functionally activeWnt3a polypeptide is between about 5 μg/μL and about 15 μg/μL, or about8 μg/μL and about 12 μg/μL. In some embodiments, the phospholipidcomprising the liposome has a tail carbon length of between about 12carbons and about 14 carbons. In some embodiments, the phospholipidcomprising the liposome has a phase transition temperature from about10° C. to about 25° C., about 15° C. to about 25° C., or about 20° C. toabout 25° C. In some embodiments, the liposome has a net charge of 0 ata pH of between about 6.5 and about 8.0, about 7.0 and about 7.8, orabout 7.2 and about 7.6. In some embodiments, the phospholipid is1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). In some embodiments,the liposome further comprises cholesterol. In some embodiments, theconcentration of DMPC and cholesterol is defined by a ratio of betweenabout 70:30 and about 100:0. In some embodiments, the Wnt3a polypeptideis mammalian Wnt3a polypeptide. In some embodiments, the mammal is ahuman. In some embodiments, the lipid modification is palmitoylation. Insome embodiments, the Wnt3a polypeptide is further glycosylated. In someembodiments, the composition is a stable composition. In someembodiments, the composition is stable up to about 106 days withoutsubstantial loss of activity. In some embodiments, the composition isstable at a temperature between about 1° C. and about 8° C. In someembodiments, the composition is stable under nitrogen.

Disclosed herein, in certain embodiments, is a composition comprising afunctionally active mammalian Wnt polypeptide and an aqueous solutioncomprising liposomes, wherein the phospholipids comprising the liposomeshave a phase transition temperature from about 10° C. to about 25° C.Also disclosed herein, in certain embodiments, is a compositioncomprising a functionally active mammalian Wnt polypeptide and anaqueous solution comprising liposomes, wherein the phospholipidscomprising the liposomes have a tail carbon length of between about 12carbons and about 14 carbons. In some embodiments, the Wnt polypeptideis integrated into the liposomal membrane. In some embodiments, the Wntpolypeptide protrudes from the liposomal membrane onto the outer surfaceof the lipid membrane. In some embodiments, the Wnt polypeptide is notincorporated into the aqueous core of the liposome. In some embodiments,the Wnt polypeptide is Wnt3a polypeptide. In some embodiments, the Wnt3apolypeptide comprises at least about 30%, about 40%, about 50%, about60%, about 70%, about 80%, about 90%, or about 95% sequence identity tothe amino acid sequence as set forth in SEQ ID NO:1. In someembodiments, the Wnt3a polypeptide comprises the amino acid sequence asset forth in SEQ ID NO:1. In some embodiments, the Wnt3a polypeptide islipid modified at an amino acid position corresponding to amino acidresidue 209 as set forth in SEQ ID NO:1 and is not lipid modified at anamino acid position corresponding to amino acid residue 77 as set forthin SEQ ID NO:1. In some embodiments, the Wnt polypeptide is Wnt5apolypeptide or Wnt10b polypeptide. In some embodiments, theconcentration of the Wnt polypeptide is between about 5 μg/μL and about15 μg/μL, or about 8 μg/μL and about 12 μg/μL. In some embodiments, theliposome has a net charge of 0 at a pH of between about 6.5 and about8.0. In some embodiments, the liposome has a net positive charge or anet negative charge at a pH of between about 6.5 and about 8.0. In someembodiments, the phospholipid is1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). In some embodiments,the liposome further comprises cholesterol. In some embodiments, theconcentration of DMPC and cholesterol is defined by a ratio of betweenabout 70:30 and about 100:0. In some embodiments, the mammal is a human.In some embodiments, the lipid modification is palmitoylation. In someembodiments, the Wnt polypeptide is further glycosylated. In someembodiments, the composition is a stable composition. In someembodiments, the composition is stable up to about 106 days withoutsubstantial loss of activity. In some embodiments, the composition isstable at a temperature between about 1° C. and about 8° C. In someembodiments, the composition is stable under nitrogen.

Disclosed herein, in certain embodiments, is a method of preparing aliposomal Wnt3a polypeptide, comprising: (a) harvesting Wnt3apolypeptides from a conditioned media comprising mammalian cells; (b)introducing the Wnt3a polypeptides to an ion-exchange column immobilizedwith a sulfonated polyaromatic compound; (c) eluting the Wnt3apolypeptides from the ion-exchange column utilizing a step gradient; and(d) contacting the Wnt3a polypeptides from step (c) with an aqueoussolution of liposomes. In some embodiments, the contacting occurs at atemperature between about 21° C. and about 25° C. In some embodiments,the time of contacting is between about 30 minutes and about 24 hours.In some embodiments, the step gradient comprises a first gradient and asecond gradient. In some embodiments, the first gradient comprises abuffer solution comprising 50 mM potassium chloride, and the secondgradient comprises a buffer solution comprising between about 150 mMpotassium chloride and about 1.5M potassium chloride. In someembodiments, the buffer solution further comprises a detergent. In someembodiments, the detergent is CHAPS or Triton X-100. In someembodiments, the conditioned media comprises up to 10% fetal bovineserum. In some embodiments, the mammalian cells are Chinese hamsterovary (CHO) cells. In some embodiments, the yield of the Wnt3apolypeptides after step (c) is between about 60% and about 90%. In someembodiments, the phospholipid comprising the liposome has a tail carbonlength of between about 12 carbons and about 14 carbons. In someembodiments, the phospholipid comprising the liposome has a phasetransition temperature from about 10° C. to about 25° C., about 15° C.to about 25° C., or about 20° C. to about 25° C. In some embodiments,the liposome has a net charge of 0 at a pH of between about 6.5 andabout 8.0, about 7.0 and about 7.8, or about 7.2 and about 7.6. In someembodiments, the phospholipid is1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). In some embodiments,the liposome further comprises cholesterol. In some embodiments, theconcentration of DMPC and cholesterol is defined by a ratio of betweenabout 70:30 and about 100:0. In some embodiments, the Wnt3a polypeptidecomprises at least about 30%, about 40%, about 50%, about 60%, about70%, about 80%, about 90%, or about 95% sequence identity to the aminoacid sequence as set forth in SEQ ID NO:1. In some embodiments, theWnt3a polypeptide comprises the amino acid sequence as set forth in SEQID NO:1. In some embodiments, the Wnt3a polypeptide is lipid modified atan amino acid position corresponding to amino acid residue 209 as setforth in SEQ ID NO:1 and is not lipid modified at an amino acid positioncorresponding to amino acid residue 77 as set forth in SEQ ID NO:1. Insome embodiments, the Wnt3a polypeptide is mammalian Wnt3a polypeptide.In some embodiments, the mammal is a human. In some embodiments, theconditioned media comprises a serum or a serum substitute. In someembodiments, the conditioned media comprises a serum. In someembodiments, the serum is a fetal bovine serum. In some embodiments, theserum concentration is between about 0.1% and about 15% in theconditioned media. In some embodiments, the conditioned media comprisesa serum substitute. In some embodiments, the serum substitute is a lipidbased substitute. In some embodiments, the conditioned media is aserum-free media. In some embodiments, the lipid modification ispalmitoylation. In some embodiments, the Wnt3a polypeptide is furtherglycosylated. In some embodiments, the Wnt3a product is separated fromthe sample mixture by centrifugation. In some embodiments, thecentrifugation time is up to 1 hour at a temperature of between about 1°C. and about 8° C. In some embodiments, the Wnt3a product aftercentrifugation is resuspended in sterile 1× phosphate buffered saline(PBS). In some embodiments, the Wnt3a product is stable under nitrogen.In some embodiments, the Wnt3a product is stable at a temperaturebetween about 1° C. and about 8° C.

Disclosed herein, in certain embodiments, is a method of preparing aliposomal Wnt polypeptide, comprising contacting a sample comprising Wntpolypeptides to an aqueous solution of liposomes, wherein thephospholipids comprising the liposomes have a phase transitiontemperature from about 10° C. to about 25° C. Also disclosed herein, incertain embodiments, is a method of preparing a liposomal Wntpolypeptide, comprising contacting a sample comprising Wnt polypeptidesto an aqueous solution of liposomes, wherein the phospholipidscomprising the liposome have a tail carbon length of between about 12carbons and about 14 carbons. In some embodiments, the contacting occursat a temperature between about 21° C. and about 25° C. In someembodiments, the time of contacting is between about 6 hours and about24 hours. In some embodiments, the method further comprises anadditional purification step prior to contacting the Wnt polypeptides toan aqueous solution of liposomes. In some embodiments, the additionalpurification step is an ion-exchange purification step, a hydrophobicpurification step, or an affinity purification step. In someembodiments, the additional purification step is an ion-exchangepurification step. In some embodiments, the ion-exchange purificationstep comprises contacting the sample with sulfonated polyaromaticcompound immobilized beads or a column immobilized with a sulfonatedpolyaromatic compound. In some embodiments, the ion-exchangepurification step comprises a step-gradient. In some embodiments, theion-exchange purification buffer comprises a detergent. In someembodiments, the detergent is CHAPS or Triton X-100. In someembodiments, the additional purification step is a hydrophobicpurification step. In some embodiments, the hydrophobic purificationstep comprises Protein A immobilized beads, or a Protein A column. Insome embodiments, the yield of the Wnt polypeptides after the additionalpurification step is between about 60% and about 99%. In someembodiments, the method further comprises harvesting Wnt polypeptidesfrom a conditioned media comprising cells from an expression cell line.In some embodiments, the expression cell line is a mammalian expressioncell line. In some embodiments, the mammalian expression cell line isChinese hamster ovary (CHO) cell line. In some embodiments, theconditioned media comprises a serum or a serum substitute. In someembodiments, the serum is a fetal bovine serum. In some embodiments, theserum concentration is between about 0.1% and about 15% in theconditioned media. In some embodiments, the conditioned media comprisesa serum substitute. In some embodiments, the serum substitute is a lipidbased substitute. In some embodiments, the conditioned media is aserum-free media. In some embodiments, the Wnt polypeptide is Wnt3apolypeptide. In some embodiments, the Wnt3a polypeptide comprises atleast about 30%, about 40%, about 50%, about 60%, about 70%, about 80%,about 90%, or about 95% sequence identity to the amino acid sequence asset forth in SEQ ID NO:1. In some embodiments, the Wnt3a polypeptidecomprises the amino acid sequence as set forth in SEQ ID NO:1. In someembodiments, the Wnt3a polypeptide is lipid modified at an amino acidposition corresponding to amino acid residue 209 as set forth in SEQ IDNO:1 and is not lipid modified at an amino acid position correspondingto amino acid residue 77 as set forth in SEQ ID NO:1. In someembodiments, the Wnt polypeptide is Wnt5a polypeptide or Wnt10bpolypeptide. In some embodiments, the liposome has a net charge of 0 ata pH of between about 6.5 and about 8.0, about 7.0 and about 7.8, orabout 7.2 and about 7.6. In some embodiments, the phospholipid is1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). In some embodiments,the liposome further comprises cholesterol. In some embodiments, theconcentration of DMPC and cholesterol is defined by a ratio of betweenabout 70:30 and about 100:0. In some embodiments, the Wnt polypeptide ismammalian Wnt polypeptide. In some embodiments, the mammal is a human.In some embodiments, the lipid modification is palmitoylation. In someembodiments, the Wnt polypeptide is further glycosylated. In someembodiments, the Wnt product is separated from the sample mixture bycentrifugation. In some embodiments, the centrifugation time is up to 1hour at a temperature of between about 1° C. and about 8° C. In someembodiments, the Wnt product after centrifugation is resuspended insterile 1×PBS. In some embodiments, the Wnt product is stable undernitrogen. In some embodiments, the Wnt product is stable at atemperature between about 1° C. and about 8° C.

Disclosed herein, in certain embodiments, is a composition of isolatedenhanced mammalian bone marrow cells wherein the cells have an enhancedexpression level in one or more of the biomarkers selected from thegroup consisting of: Runx2, Osterix, Osteocalcin, Osteopontin, alkalinephosphatase, collagen type I, Axin2, Lef1, and Tcf4, after treatmentwith a liposomal Wnt polypeptide. In some embodiments, the mammalianbone marrow cells are rodent bone marrow cells. In some embodiments, therodent bone marrow cells are harvested from a rodent that is older than10 months. In some embodiments, the mammalian bone marrow cells arehuman bone marrow cells. In some embodiments, the human bone marrowcells are obtained from a subject at or older than 35 years of age. Insome embodiments, the biomarkers are selected from Osteocalcin,Osteopontin, Axin2, Lef1, and Tcf4. In some embodiments, the biomarkersare selected from Osteocalcin, and Osteopontin. In some embodiments, theenhanced expression level is compared to untreated mammalian bone marrowcells. In some embodiments, the enhanced expression level is betweenabout 2% and about 20% or about 4% and about 10% compared to untreatedmammalian bone marrow cells. In some embodiments, the mammalian bonemarrow cells further have a decreased expression level in a biomarkerselected from SOX9 and PPARγ after treatment with liposomal Wntpolypeptide. In some embodiments, the decreased expression level in themammalian bone marrow cells are compared to untreated mammalian bonemarrow cells. In some embodiments, the bone marrow cells furthercomprises a decrease in apoptosis level compared to untreated mammalianbone marrow cells. In some embodiments, the decrease in apoptosis levelis about 50%. In some embodiments, the mammalian bone marrow cellscomprise enhanced osteogenic potential compared to untreated mammalianbone marrow cells. In some embodiments, the enhanced osteogenicpotential comprises new bone growth level at the site of bone defectafter transplanting treated mammalian bone marrow cells. In someembodiments, the new bone growth level is compared to new bone growthlevel of transplanted untreated mammalian bone marrow cells. In someembodiments, the new bone growth level of transplanted treated mammalianbone marrow cells is between about 1% and about 20% or about 5% andabout 12% compared to the new bone growth level of transplanteduntreated mammalian bone marrow cells. In some embodiments, themammalian bone marrow cells include adherent and non-adherent bonemarrow cells. In some embodiments, the mammalian bone marrow cells areadherent bone marrow cells. In some embodiments, the adherent bonemarrow cells include bone marrow stem cells and bone marrow progenitorcells. In some embodiments, the adherent bone marrow cells include bonemarrow stromal cells. In some embodiments, the mammalian bone marrowcells are autologous bone marrow cells. In some embodiments, theautologous bone marrow cells include adherent and non-adherent bonemarrow cells. In some embodiments, the autologous bone marrow cells areadherent bone marrow cells. In some embodiments, the autologous bonemarrow cells include bone marrow stem cells and bone marrow progenitorcells. In some embodiments, the autologous bone marrow cells includebone marrow stromal cells. In some embodiments, the mammalian bonemarrow cells are allogeneic bone marrow cells. In some embodiments, theallogeneic bone marrow cells include adherent and non-adherent bonemarrow cells. In some embodiments, the allogeneic bone marrow cells areadherent bone marrow cells. In some embodiments, the allogeneic bonemarrow cells include bone marrow stem cells and bone marrow progenitorcells. In some embodiments, the allogeneic bone marrow cells includebone marrow stromal cells. In some embodiments, the human bone marrowcells post treatment with liposomal Wnt polypeptide exhibit biomarkerexpression levels that are observed in a human subject younger than 35years of age. In some embodiments, the liposomal Wnt polypeptidecomprises Wnt polypeptides and an aqueous solution of liposomes. In someembodiments, the phospholipids comprising the liposomes have a phasetransition temperature from about 10° C. to about 25° C. In someembodiments, the phospholipids comprising the liposomes have a tailcarbon length of between about 12 carbons and about 14 carbons. In someembodiments, the Wnt polypeptide is Wnt3a polypeptide. In someembodiments, the Wnt3a polypeptide comprises at least about 30%, about40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 95%sequence identity to the amino acid sequence as set forth in SEQ IDNO:1. In some embodiments, the Wnt3a polypeptide comprises the aminoacid sequence as set forth in SEQ ID NO:1. In some embodiments, theWnt3a polypeptide is lipid modified at an amino acid positioncorresponding to amino acid residue 209 as set forth in SEQ ID NO:1, andis not lipid modified at an amino acid position corresponding to aminoacid residue 77 as set forth in SEQ ID NO:1. In some embodiments, theWnt polypeptide is Wnt5a polypeptide or Wnt10b polypeptide.

Disclosed herein, in certain embodiments, is a composition of mammalianbone marrow cells obtained from a human subject at or older than 35years of age, wherein the mammalian bone marrow cells express anenhanced expression level in one or more of the biomarkers selected fromthe group consisting of: Runx2, Osterix, Osteocalcin, Osteopontin,alkaline phosphatase, collagen type I, Axin2, Lef1, and Tcf4, aftertreatment with a liposomal Wnt polypeptide. In some embodiments, thebiomarkers are selected from Osteocalcin, Osteopontin, Axin2, Lef1, andTcf4. In some embodiments, the biomarkers are selected from Osteocalcin,and Osteopontin. In some embodiments, the enhanced expression level iscompared to untreated mammalian bone marrow cells. In some embodiments,the enhanced expression level is between about 2% and about 20% or about4% and about 10% compared to untreated mammalian bone marrow cells. Insome embodiments, the mammalian bone marrow cells further have adecreased expression level in a biomarker selected from SOX9 and PPARγafter treatment with liposomal Wnt polypeptide. In some embodiments, thedecreased expression level in the mammalian bone marrow cells arecompared to untreated mammalian bone marrow cells. In some embodiments,the bone marrow cells further comprises a decrease in apoptosis levelcompared to untreated mammalian bone marrow cells. In some embodiments,the decrease in apoptosis level is about 50%. In some embodiments, themammalian bone marrow cells comprise enhanced osteogenic potentialcompared to untreated mammalian bone marrow cells. In some embodiments,the enhanced osteogenic potential comprises new bone growth level at thesite of bone defect after transplanting treated mammalian bone marrowcells. In some embodiments, the new bone growth level is compared to newbone growth level of transplanted untreated mammalian bone marrow cells.In some embodiments, the new bone growth level of transplanted treatedmammalian bone marrow cells is between about 1% and about 20% or about5% and about 12% compared to the new bone growth level of transplanteduntreated mammalian bone marrow cells. In some embodiments, themammalian bone marrow cells include adherent and non-adherent bonemarrow cells. In some embodiments, the mammalian bone marrow cells areadherent bone marrow cells. In some embodiments, the adherent bonemarrow cells include bone marrow stem cells and bone marrow progenitorcells. In some embodiments, the adherent bone marrow cells include bonemarrow stromal cells. In some embodiments, the mammalian bone marrowcells are autologous bone marrow cells. In some embodiments, theautologous bone marrow cells include adherent and non-adherent bonemarrow cells. In some embodiments, the autologous bone marrow cells areadherent bone marrow cells. In some embodiments, the autologous bonemarrow cells include bone marrow stem cells and bone marrow progenitorcells. In some embodiments, the autologous bone marrow cells includebone marrow stromal cells. In some embodiments, the mammalian bonemarrow cells are allogeneic bone marrow cells. In some embodiments, theallogeneic bone marrow cells include adherent and non-adherent bonemarrow cells. In some embodiments, the allogeneic bone marrow cells areadherent bone marrow cells. In some embodiments, the allogeneic bonemarrow cells include bone marrow stem cells and bone marrow progenitorcells. In some embodiments, the allogeneic bone marrow cells includebone marrow stromal cells. In some embodiments, the human bone marrowcells post treatment with liposomal Wnt polypeptide exhibit biomarkerexpression levels that are observed in a human subject younger than 35years of age. In some embodiments, the liposomal Wnt polypeptidecomprises Wnt polypeptides and an aqueous solution of liposomes. In someembodiments, the phospholipids comprising the liposomes have a phasetransition temperature from about 10° C. to about 25° C. In someembodiments, the phospholipids comprising the liposomes have a tailcarbon length of between about 12 carbons and about 14 carbons. In someembodiments, the Wnt polypeptide is Wnt3a polypeptide. In someembodiments, the Wnt3a polypeptide comprises at least about 30%, about40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 95%sequence identity to the amino acid sequence as set forth in SEQ IDNO:1. In some embodiments, the Wnt3a polypeptide comprises the aminoacid sequence as set forth in SEQ ID NO:1. In some embodiments, theWnt3a polypeptide is lipid modified at an amino acid positioncorresponding to amino acid residue 209 as set forth in SEQ ID NO:1, andis not lipid modified at an amino acid position corresponding to aminoacid residue 77 as set forth in SEQ ID NO:1. In some embodiments, theWnt polypeptide is Wnt5a polypeptide or Wnt10b polypeptide.

Disclosed herein, in certain embodiments, is a mammalian bone marrowcomposition produced by a process comprising contacting isolatedmammalian bone marrow cells ex-vivo with a liposomal Wnt polypeptide,wherein the contacting time is between about 30 minutes and about 4hours. In some embodiments, the contacting temperature is at betweenabout 0° C. and about 37° C., or about 20° C., and about 25° C. In someembodiments, the process further comprises a washing step aftercontacting to remove free liposomal Wnt polypeptide. In someembodiments, the process further comprises transplanting the liposomalWnt polypeptide to a site of bone defect. In some embodiments, the bonemarrow cells have an enhanced expression level in one or more of thebiomarkers selected from the group consisting of: Runx2, Osterix,Osteocalcin, Osteopontin, alkaline phosphatase, collagen type I, Axin2,Lef1, and Tcf4, compared to untreated mammalian bone marrow cells. Insome embodiments, the biomarkers are selected from Osteocalcin,Osteopontin, Axin2, Lef1, and Tcf4. In some embodiments, the biomarkersare selected from Osteocalcin, and Osteopontin. In some embodiments, theenhanced expression level is between about 2% and about 20% or about 4%and about 10% compared to untreated mammalian bone marrow cells. In someembodiments, the mammalian bone marrow cells further have a decreasedexpression level in a biomarker selected from SOX9 and PPARγ aftertreatment with liposomal Wnt polypeptide. In some embodiments, thedecreased expression level in the mammalian bone marrow cells arecompared to untreated mammalian bone marrow cells. In some embodiments,the bone marrow cells further comprises decrease in apoptosis levelcompared to untreated mammalian bone marrow cells. In some embodiments,the decrease in apoptosis level is about 50%. In some embodiments, themammalian bone marrow cells comprise enhanced osteogenic potentialcompared to untreated mammalian bone marrow cells. In some embodiments,the enhanced osteogenic potential comprises new bone growth level at thesite of bone defect after transplanting treated mammalian bone marrowcells. In some embodiments, the new bone growth level is compared to newbone growth level of transplanted untreated mammalian bone marrow cells.In some embodiments, the new bone growth level of transplanted treatedmammalian bone marrow cells is between about 1% and about 20% or about5% and about 12% compared to the new bone growth level of transplanteduntreated mammalian bone marrow cells. In some embodiments, themammalian bone marrow cells include adherent and non-adherent bonemarrow cells. In some embodiments, the mammalian bone marrow cells areadherent bone marrow cells. In some embodiments, the adherent bonemarrow cells include bone marrow stem cells and bone marrow progenitorcells. In some embodiments, the adherent bone marrow cells include bonemarrow stromal cells. In some embodiments, the mammalian bone marrowcells are autologous bone marrow cells. In some embodiments, theautologous bone marrow cells are harvested from femurs, tibiae, and/oriliac crest. In some embodiments, the autologous bone marrow cellsinclude adherent and non-adherent bone marrow cells. In someembodiments, the autologous bone marrow cells are adherent bone marrowcells. In some embodiments, the autologous bone marrow cells includebone marrow stem cells and bone marrow progenitor cells. In someembodiments, the autologous bone marrow cells include bone marrowstromal cells. In some embodiments, the isolated mammalian bone marrowcells are allogeneic bone marrow cells. In some embodiments, theallogeneic bone marrow cells include adherent and non-adherent bonemarrow cells. In some embodiments, the allogeneic bone marrow cells areadherent bone marrow cells. In some embodiments, the allogeneic bonemarrow cells include bone marrow stem cells and bone marrow progenitorcells. In some embodiments, the allogeneic bone marrow cells includebone marrow stromal cells. In some embodiments, the mammalian bonemarrow cells are rodent bone marrow cells. In some embodiments, therodent bone marrow cells are harvested from a rodent that is older than10 months. In some embodiments, the mammalian bone marrow cells arehuman bone marrow cells. In some embodiments, the human bone marrowcells are harvested from a subject at or older than 35 years of age. Insome embodiments, the human bone marrow cells post treatment withliposomal Wnt polypeptide exhibit biomarker expression levels that areobserved in a subject younger than 35 years of age. In some embodiments,the liposomal Wnt polypeptide comprises Wnt polypeptides and an aqueoussolution of liposomes. In some embodiments, the phospholipids comprisingthe liposomes have a phase transition temperature from about 10° C. toabout 25° C. In some embodiments, the phospholipids comprising theliposomes have a tail carbon length of between about 12 carbons andabout 14 carbons. In some embodiments, the Wnt polypeptide is Wnt3apolypeptide. In some embodiments, the Wnt3a polypeptide comprises atleast about 30%, about 40%, about 50%, about 60%, about 70%, about 80%,about 90%, or about 95% sequence identity to the amino acid sequence asset forth in SEQ ID NO:1. In some embodiments, the Wnt3a polypeptidecomprises the amino acid sequence as set forth in SEQ ID NO:1. In someembodiments, the Wnt3a polypeptide is lipid modified at an amino acidposition corresponding to amino acid residue 209 as set forth in SEQ IDNO:1 and is not lipid modified at an amino acid position correspondingto amino acid residue 77 as set forth in SEQ ID NO:1. In someembodiments, the Wnt polypeptide is Wnt5a polypeptide or Wnt10bpolypeptide.

Disclosed herein, in certain embodiments, is a method of treating a bonedefect in a subject, comprising: (a) contacting a sample comprisingmammalian bone marrow cells ex-vivo with a liposomal Wnt3a polypeptide;(b) washing the sample to remove free liposomal Wnt3a polypeptide; and(c) transplanting the liposomal Wnt3a polypeptide treated bone graftmaterials into a site of bone defect. In some embodiments, thecontacting temperature is at between about 0° C. and about 37° C., orabout 20° C. and about 25° C. In some embodiments, the contacting timeis between about 30 minutes and about 4 hours. In some embodiments, thebone marrow cells have an enhanced expression level in one or more ofthe biomarkers selected from the group consisting of: Runx2, Osterix,Osteocalcin, Osteopontin, alkaline phosphatase, collagen type I, Axin2,Lef1, and Tcf4, compared to untreated mammalian bone marrow cells. Insome embodiments, the biomarkers are selected from Osteocalcin,Osteopontin, Axin2, Lef1, and Tcf4. In some embodiments, the biomarkersare selected from Osteocalcin, and Osteopontin. In some embodiments, theenhanced expression level is between about 2% and about 20% or about 4%and about 10% compared to untreated mammalian bone marrow cells. In someembodiments, the mammalian bone marrow cells further have a decreasedexpression level in a biomarker selected from SOX9 and PPARγ aftertreatment with liposomal Wnt3a polypeptide. In some embodiments, thedecreased expression level in the mammalian bone marrow cells arecompared to untreated mammalian bone marrow cells. In some embodiments,the bone marrow cells further comprises decrease in apoptosis levelcompared to untreated mammalian bone marrow cells. In some embodiments,the decrease in apoptosis level is about 50%. In some embodiments, themammalian bone marrow cells comprise enhanced osteogenic potentialcompared to untreated mammalian bone marrow cells. In some embodiments,the enhanced osteogenic potential comprises new bone growth level at thesite of bone defect after transplanting treated mammalian bone marrowcells. In some embodiments, the new bone growth level is compared to newbone growth level of transplanted untreated mammalian bone marrow cells.In some embodiments, the new bone growth level of transplanted treatedmammalian bone marrow cells is between about 1% and about 20% or about5% and about 12% compared to the new bone growth level of transplanteduntreated mammalian bone marrow cells. In some embodiments, themammalian bone marrow cells include adherent and non-adherent bonemarrow cells. In some embodiments, the mammalian bone marrow cells areadherent bone marrow cells. In some embodiments, the adherent bonemarrow cells include bone marrow stem cells and bone marrow progenitorcells. In some embodiments, the adherent bone marrow cells include bonemarrow stromal cells. In some embodiments, the mammalian bone marrowcells are autologous bone marrow cells. In some embodiments, theautologous bone marrow cells are harvested from femurs, tibiae, and/oriliac crest. In some embodiments, the autologous bone marrow cellsinclude adherent and non-adherent bone marrow cells. In someembodiments, the autologous bone marrow cells are adherent bone marrowcells. In some embodiments, the autologous bone marrow cells includebone marrow stem cells and bone marrow progenitor cells. In someembodiments, the autologous bone marrow cells include bone marrowstromal cells. In some embodiments, the isolated mammalian bone marrowcells are allogeneic bone marrow cells. In some embodiments, theallogeneic bone marrow cells include adherent and non-adherent bonemarrow cells. In some embodiments, the allogeneic bone marrow cells areadherent bone marrow cells. In some embodiments, the allogeneic bonemarrow cells include bone marrow stem cells and bone marrow progenitorcells. In some embodiments, the allogeneic bone marrow cells includebone marrow stromal cells. In some embodiments, the mammalian bonemarrow cells are rodent bone marrow cells. In some embodiments, therodent bone marrow cells are harvested from a rodent that is older than10 months. In some embodiments, the mammalian bone marrow cells arehuman bone marrow cells. In some embodiments, the human bone marrowcells are harvested from a subject at or older than 35 years of age. Insome embodiments, the human bone marrow cells post treatment withliposomal Wnt3a polypeptide exhibit biomarker expression levels that areobserved in a subject younger than 35 years of age. In some embodiments,the liposomal Wnt3a polypeptide comprises Wnt3a polypeptides and anaqueous solution of liposomes. In some embodiments, the phospholipidscomprising the liposomes have a phase transition temperature from about10° C. to about 25° C. In some embodiments, the phospholipids comprisingthe liposomes have a tail carbon length of between about 12 carbons andabout 14 carbons. In some embodiments, the Wnt3a polypeptide comprisesat least about 30%, about 40%, about 50%, about 60%, about 70%, about80%, about 90%, or about 95% sequence identity to the amino acidsequence as set forth in SEQ ID NO:1. In some embodiments, the Wnt3apolypeptide comprises the amino acid sequence as set forth in SEQ IDNO:1. In some embodiments, the Wnt3a polypeptide is lipid modified at anamino acid position corresponding to amino acid residue 209 as set forthin SEQ ID NO:1 and is not lipid modified at an amino acid positioncorresponding to amino acid residue 77 as set forth in SEQ ID NO:1.

Disclosed herein, in certain embodiments, is a method of treating a bonedefect in a subject, comprising: (a) contacting a sample comprisingmammalian bone marrow cells ex-vivo with a liposomal Wnt polypeptide;(b) washing the sample to remove free liposomal Wnt polypeptide; and (c)transplanting the liposomal Wnt polypeptide treated bone graft materialsinto a site of bone defect. In some embodiments, the contactingtemperature is at between about 0° C. and about 37° C. or about 20° C.and about 25° C. In some embodiments, the contacting time is betweenabout 30 minutes and about 4 hours. In some embodiments, the bone marrowcells have an enhanced expression level in one or more of the biomarkersselected from the group consisting of: Runx2, Osterix, Osteocalcin,Osteopontin, alkaline phosphatase, collagen type I, Axin2, Lef1, andTcf4, compared to untreated mammalian bone marrow cells. In someembodiments, the biomarkers are selected from Osteocalcin, Osteopontin,Axin2, Lef1, and Tcf4. In some embodiments, the biomarkers are selectedfrom Osteocalcin, and Osteopontin. In some embodiments, the enhancedexpression level is between about 2% and about 20% or about 4% and about10% compared to untreated mammalian bone marrow cells. In someembodiments, the mammalian bone marrow cells further have a decreasedexpression level in a biomarker selected from SOX9 and PPARγ aftertreatment with liposomal Wnt polypeptide. In some embodiments, thedecreased expression level in the mammalian bone marrow cells arecompared to untreated mammalian bone marrow cells. In some embodiments,the bone marrow cells further comprises decrease in apoptosis levelcompared to untreated mammalian bone marrow cells. In some embodiments,the decrease in apoptosis level is about 50%. In some embodiments, themammalian bone marrow cells comprise enhanced osteogenic potentialcompared to untreated mammalian bone marrow cells. In some embodiments,the enhanced osteogenic potential comprises new bone growth level at thesite of bone defect after transplanting treated mammalian bone marrowcells. In some embodiments, the new bone growth level is compared to newbone growth level of transplanted untreated mammalian bone marrow cells.In some embodiments, the new bone growth level of transplanted treatedmammalian bone marrow cells is between about 1% and about 20% or about5% and about 12% compared to the new bone growth level of transplanteduntreated mammalian bone marrow cells. In some embodiments, themammalian bone marrow cells include adherent and non-adherent bonemarrow cells. In some embodiments, the mammalian bone marrow cells areadherent bone marrow cells. In some embodiments, the adherent bonemarrow cells include bone marrow stem cells and bone marrow progenitorcells. In some embodiments, the adherent bone marrow cells include bonemarrow stromal cells. In some embodiments, the mammalian bone marrowcells are autologous bone marrow cells. In some embodiments, theautologous bone marrow cells are harvested from femurs, tibiae, and/oriliac crest. In some embodiments, the autologous bone marrow cellsinclude adherent and non-adherent bone marrow cells. In someembodiments, the autologous bone marrow cells are adherent bone marrowcells. In some embodiments, the autologous bone marrow cells includebone marrow stem cells and bone marrow progenitor cells. In someembodiments, the autologous bone marrow cells include bone marrowstromal cells. In some embodiments, the isolated mammalian bone marrowcells are allogeneic bone marrow cells. In some embodiments, theallogeneic bone marrow cells include adherent and non-adherent bonemarrow cells. In some embodiments, the allogeneic bone marrow cells areadherent bone marrow cells. In some embodiments, the allogeneic bonemarrow cells include bone marrow stem cells and bone marrow progenitorcells. In some embodiments, the allogeneic bone marrow cells includebone marrow stromal cells. In some embodiments, the mammalian bonemarrow cells are rodent bone marrow cells. In some embodiments, therodent bone marrow cells are harvested from a rodent that is older than10 months. In some embodiments, the mammalian bone marrow cells arehuman bone marrow cells. In some embodiments, the human bone marrowcells are harvested from a subject at or older than 35 years of age. Insome embodiments, the human bone marrow cells post treatment withliposomal Wnt polypeptide exhibit biomarker expression levels that areobserved in a subject younger than 35 years of age. In some embodiments,the liposomal Wnt polypeptide comprises Wnt polypeptides and an aqueoussolution of liposomes. In some embodiments, the phospholipids comprisingthe liposomes have a phase transition temperature from about 10° C. toabout 25° C. In some embodiments, the phospholipids comprising theliposomes have a tail carbon length of between about 12 carbons andabout 14 carbons. In some embodiments, the Wnt polypeptide is Wnt3apolypeptide. In some embodiments, the Wnt3a polypeptide comprises atleast about 30%, about 40%, about 50%, about 60%, about 70%, about 80%,about 90%, or about 95% sequence identity to the amino acid sequence asset forth in SEQ ID NO:1. In some embodiments, the Wnt3a polypeptidecomprises the amino acid sequence as set forth in SEQ ID NO:1. In someembodiments, the Wnt3a polypeptide is lipid modified at an amino acidposition corresponding to amino acid residue 209 as set forth in SEQ IDNO:1 and is not lipid modified at an amino acid position correspondingto amino acid residue 77 as set forth in SEQ ID NO:1. In someembodiments, the Wnt polypeptide is Wnt5a polypeptide or Wnt10bpolypeptide.

Disclosed herein, in certain embodiments, is a kit for generating bonegraft materials comprising the liposomal Wnt polypeptide. In someembodiments, the liposomal Wnt polypeptide is a composition comprising afunctionally active mammalian Wnt polypeptide and an aqueous solutioncomprising liposomes, wherein the phospholipids comprising the liposomeshave a phase transition temperature from about 10° C. to about 25° C. Insome embodiments, the liposomal Wnt polypeptide is a compositioncomprising a functionally active mammalian Wnt polypeptide and anaqueous solution comprising liposomes, wherein the phospholipidscomprising the liposomes have a tail carbon length of between about 12carbons and about 14 carbons. In some embodiments, the Wnt polypeptideis integrated into the liposomal membrane. In some embodiments, the Wntpolypeptide protrudes from the liposomal membrane onto the outer surfaceof the lipid membrane. In some embodiments, the Wnt polypeptide is notincorporated into the aqueous core of the liposome. In some embodiments,the Wnt polypeptide is Wnt3a polypeptide. In some embodiments, the Wnt3apolypeptide comprises at least about 30%, about 40%, about 50%, about60%, about 70%, about 80%, about 90%, or about 95% sequence identity tothe amino acid sequence as set forth in SEQ ID NO:1. In someembodiments, the Wnt3a polypeptide comprises the amino acid sequence asset forth in SEQ ID NO:1. In some embodiments, the Wnt3a polypeptide islipid modified at an amino acid position corresponding to amino acidresidue 209 as set forth in SEQ ID NO:1 and is not lipid modified at anamino acid position corresponding to amino acid residue 77 as set forthin SEQ ID NO:1. In some embodiments, the Wnt polypeptide is Wnt5apolypeptide or Wnt10b polypeptide. In some embodiments, theconcentration of the Wnt polypeptide is between about 5 μg/μL and about15 μg/μL, or about 8 μg/μL and about 12 μg/μL. In some embodiments, theliposome has a net charge of 0 at a pH of between about 6.5 and about8.0. In some embodiments, the liposome has a net positive charge or anet negative charge at a pH of between about 6.5 and about 8.0. In someembodiments, the phospholipid is1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). In some embodiments,the liposome further comprises cholesterol. In some embodiments, theconcentration of DMPC and cholesterol is defined by a ratio of betweenabout 70:30 and about 100:0. In some embodiments, the mammal is a human.In some embodiments, the lipid modification is palmitoylation. In someembodiments, the Wnt polypeptide is further glycosylated. In someembodiments, the composition is a stable composition. In someembodiments, the composition is stable up to about 106 days withoutsubstantial loss of activity. In some embodiments, the composition isstable at a temperature between about 1° C. and about 8° C. In someembodiments, the composition is stable under nitrogen.

Disclosed herein, in certain embodiments, is a kit for generating bonegraft materials comprising the liposomal Wnt3a polypeptide. In someembodiments, the liposomal Wnt3a polypeptide is a composition comprisinga functionally active Wnt3a polypeptide and an aqueous solutioncomprising liposomes, wherein the functionally active Wnt3a polypeptidecomprises a lipid modification at an amino acid position correspondingto amino acid residue 209 as set forth in SEQ ID NO:1. In someembodiments, the functionally active Wnt3a polypeptide is not lipidmodified at an amino acid position corresponding to amino acid residue77 as set forth in SEQ ID NO:1. In some embodiments, the functionallyactive Wnt3a polypeptide is integrated into the liposomal membrane. Insome embodiments, the functionally active Wnt3a polypeptide protrudesfrom the liposomal membrane onto the outer surface of the lipidmembrane. In some embodiments, the functionally active Wnt3a polypeptideis not incorporated into the aqueous core of the liposome. In someembodiments, the functionally active Wnt3a polypeptide comprises atleast about 30%, about 40%, about 50%, about 60%, about 70%, about 80%,about 90%, or about 95% sequence identity to the amino acid sequence asset forth in SEQ ID NO:1. In some embodiments, the functionally activeWnt3a polypeptide comprises the amino acid sequence as set forth in SEQID NO:1. In some embodiments, the concentration of the functionallyactive Wnt3a polypeptide is between about 5 μg/μL and about 15 μg/μL, orabout 8 μg/μL and about 12 μg/μL. In some embodiments, the phospholipidcomprising the liposome has a tail carbon length of between about 12carbons and about 14 carbons. In some embodiments, the phospholipidcomprising the liposome has a phase transition temperature from about10° C. to about 25° C., about 15° C. to about 25° C., or about 20° C. toabout 25° C. In some embodiments, the liposome has a net charge of 0 ata pH of between about 6.5 and about 8.0, about 7.0 and about 7.8, orabout 7.2 and about 7.6. In some embodiments, the phospholipid is1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). In some embodiments,the liposome further comprises cholesterol. In some embodiments, theconcentration of DMPC and cholesterol is defined by a ratio of betweenabout 70:30 and about 100:0. In some embodiments, the Wnt3a polypeptideis mammalian Wnt3a polypeptide. In some embodiments, the mammal is ahuman. In some embodiments, the lipid modification is palmitoylation. Insome embodiments, the Wnt3a polypeptide is further glycosylated. In someembodiments, the composition is a stable composition. In someembodiments, the composition is stable up to about 106 days withoutsubstantial loss of activity. In some embodiments, the composition isstable at a temperature between about 1° C. and about 8° C. In someembodiments, the composition is stable under nitrogen.

Disclosed herein, in certain embodiments, is a kit for generating bonegraft materials comprising an isolated enhanced mammalian bone marrowcell composition. In some embodiments, the isolated enhanced mammalianbone marrow cell composition is a composition of mammalian bone marrowcells obtained from a human subject at or older than 35 years of age,wherein the mammalian bone marrow cells express an enhanced expressionlevel in one or more of the biomarkers selected from the groupconsisting of: Runx2, Osterix, Osteocalcin, Osteopontin, alkalinephosphatase, collagen type I, Axin2, Lef1, and Tcf4, after treatmentwith a liposomal Wnt polypeptide. In some embodiments, the biomarkersare selected from Osteocalcin, Osteopontin, Axin2, Lef1, and Tcf4. Insome embodiments, the biomarkers are selected from Osteocalcin, andOsteopontin. In some embodiments, the enhanced expression level iscompared to untreated mammalian bone marrow cells. In some embodiments,the enhanced expression level is between about 2% and about 20% or about4% and about 10% compared to untreated mammalian bone marrow cells. Insome embodiments, the mammalian bone marrow cells further have adecreased expression level in a biomarker selected from SOX9 and PPARγafter treatment with liposomal Wnt polypeptide. In some embodiments, thedecreased expression level in the mammalian bone marrow cells arecompared to untreated mammalian bone marrow cells. In some embodiments,the bone marrow cells further comprises a decrease in apoptosis levelcompared to untreated mammalian bone marrow cells. In some embodiments,the decrease in apoptosis level is about 50%. In some embodiments, themammalian bone marrow cells comprise enhanced osteogenic potentialcompared to untreated mammalian bone marrow cells. In some embodiments,the enhanced osteogenic potential comprises new bone growth level at thesite of bone defect after transplanting treated mammalian bone marrowcells. In some embodiments, the new bone growth level is compared to newbone growth level of transplanted untreated mammalian bone marrow cells.In some embodiments, the new bone growth level of transplanted treatedmammalian bone marrow cells is between about 1% and about 20% or about5% and about 12% compared to the new bone growth level of transplanteduntreated mammalian bone marrow cells. In some embodiments, themammalian bone marrow cells include adherent and non-adherent bonemarrow cells. In some embodiments, the mammalian bone marrow cells areadherent bone marrow cells. In some embodiments, the adherent bonemarrow cells include bone marrow stem cells and bone marrow progenitorcells. In some embodiments, the adherent bone marrow cells include bonemarrow stromal cells. In some embodiments, the mammalian bone marrowcells are autologous bone marrow cells. In some embodiments, theautologous bone marrow cells include adherent and non-adherent bonemarrow cells. In some embodiments, the autologous bone marrow cells areadherent bone marrow cells. In some embodiments, the autologous bonemarrow cells include bone marrow stem cells and bone marrow progenitorcells. In some embodiments, the autologous bone marrow cells includebone marrow stromal cells. In some embodiments, the mammalian bonemarrow cells are allogeneic bone marrow cells. In some embodiments, theallogeneic bone marrow cells include adherent and non-adherent bonemarrow cells. In some embodiments, the allogeneic bone marrow cells areadherent bone marrow cells. In some embodiments, the allogeneic bonemarrow cells include bone marrow stem cells and bone marrow progenitorcells. In some embodiments, the allogeneic bone marrow cells includebone marrow stromal cells. In some embodiments, the human bone marrowcells post treatment with liposomal Wnt polypeptide exhibit biomarkerexpression levels that are observed in a human subject younger than 35years of age. In some embodiments, the liposomal Wnt polypeptidecomprises Wnt polypeptides and an aqueous solution of liposomes. In someembodiments, the phospholipids comprising the liposomes have a phasetransition temperature from about 10° C. to about 25° C. In someembodiments, the phospholipids comprising the liposomes have a tailcarbon length of between about 12 carbons and about 14 carbons. In someembodiments, the Wnt polypeptide is Wnt3a polypeptide. In someembodiments, the Wnt3a polypeptide comprises at least about 30%, about40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 95%sequence identity to the amino acid sequence as set forth in SEQ IDNO:1. In some embodiments, the Wnt3a polypeptide comprises the aminoacid sequence as set forth in SEQ ID NO:1. In some embodiments, theWnt3a polypeptide is lipid modified at an amino acid positioncorresponding to amino acid residue 209 as set forth in SEQ ID NO:1, andis not lipid modified at an amino acid position corresponding to aminoacid residue 77 as set forth in SEQ ID NO:1. In some embodiments, theWnt polypeptide is Wnt5a polypeptide or Wnt10b polypeptide.

Further aspects and embodiment will be apparent from the rest of thedisclosure, and are included within the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is best understood from the following detailed descriptionwhen read in conjunction with the accompanying drawings. The patent orapplication file contains at least one drawing executed in color. Copiesof this patent or patent application publication with color drawing(s)will be provided by the Office upon request and payment of the necessaryfee. It is emphasized that, according to common practice, the variousfeatures of the drawings are not to-scale. On the contrary, thedimensions of the various features are arbitrarily expanded or reducedfor clarity. Included in the drawings are the following figures.

FIG. 1 illustrates a purification scheme of the present invention.

FIG. 2A-2B indicates SDS PAGE and Coomassie blue staining analyses ofWnt3a.

FIG. 3A-3D exemplifies a mass spectroscopy data.

FIG. 4A-4B illustrates TEM characterization of L-Wnt3a. TEM was used tovisualize L-Wnt3a, and the average diameter of the vesicles wascalculated.

FIG. 5A-5B illustrates quantification of human Wnt3a and L-Wnt3aactivities. Bioluminescence was quantified with triplicate reads on adual-light ready luminometer (Berthold). Activity of Human Wnt3a (ng/uL;FIG. 5A) and L-Wnt3a was defined (FIG. 5B) from a standard curvegenerated by serial dilutions of Wnt3a protein.

FIG. 6A-6B indicates the stability of L-Wnt3a at 4° C. Theliposome-Wnt3a interaction was examined. Liposomal Wnt3a (L-Wnt3a) wasincubated at 4° C. for up to 48 days. Human L-Wnt3a polypeptide was thentested in a LSL assay for activity. L-Wnt3a polypeptide showed no lossof activity over the 48-day test period.

FIG. 7A-7B indicates the stability of L-Wnt3a at 23° C.

FIG. 8A-8C illustrates the stability of L-Wnt3a at 37° C. Theassociation of human Wnt3a with a DMPC:cholesterol liposome was testedto determine whether it would provide stability to Wnt3a polypeptide.Data from multiple time points were fit to a single exponential decay,which showed that after 10 h, L-Wnt3a retained half of its activity at37° C. (blue line, FIG. 8A) whereas Wnt3a lost its activity within 5 min(red line, FIG. 8A). The loss of activity in human Wnt3a was due toproteolysis. In Wnt3a+CHAPS solutions, smaller molecular weight bandswere detectable in the immunoblots (FIG. 8B). An immunoblot of L-Wnt3apreparations did not detect smaller molecular weight bands correspondingto Wnt3a (FIG. 8C).

FIG. 9 exemplifies human Wnt3a polypeptide produced from cells grown insuspension versus adherent cultures.

FIG. 10 illustrates doxycycline concentration-dependent induction ofhuman Wnt3a polypeptide secretion. Doxycycline was added to the media ata range of concentrations (ng/uL), which influenced the amount of humanWnt3a produced by CHO cells.

FIG. 11 exemplifies that fetal bovine serum was used for human Wnt3apolypeptide secretion from CHO cells.

FIG. 12 illustrates human Wnt3a secretion from CHO cells as a functionof days in culture.

FIG. 13A-130 illustrates robustness, precision, limit of detection andspecificity of the LSL cellular reporter assay.

FIG. 14A-14B illustrates L-Wnt3a dose response curves using primarycells. (FIG. 14A) In mouse embryonic fibroblasts (MEFs), the linearrange of effective concentration was 0.025-0.1 ng/μL human Wnt3a. (FIG.14B) L-Wnt3a activity in bone marrow-derived stem cells, and determinedthat the linear range of effective concentrations ranged from 0.004-0.08ng/μL.

FIG. 15 illustrates human Wnt3a activity under different lipidformulations. DMPC and cholesterol lipids were substituted for CHAPS tomaintain Wnt3a activity but liposomes fabricated with lipids such asMPPC, DPPC, DMPS, DMPG, and DMGE were inactive.

FIG. 16A-16E exemplifies a sucrose density gradient.

FIG. 17A-170 exemplifies human Wnt3a affinity toward liposome membranes.The Wnt3a/Frizzled binding affinity was about 3.6 nM. The Wnt3a/LRP6binding affinity was about 9 nM. The binding affinity between Wnt3a andthe liposomal membrane was about ˜6 nM, based on pull-down assaysbetween Wnt3a, Frizzled (Fz), and Lrp6.

FIG. 18A-180 illustrates kinetics of human Wnt3a association withliposomes.

FIG. 19 illustrates Wnt5a and Wnt10b polypeptide bindings to neutralDMPC liposomes.

FIG. 20A-20L illustrates individual components of the bone graftmaterial. The bone graft material contains stem and progenitor cellpopulations. FIG. 20A indicates Gomori staining of BGM harvested fromrat femur, (FIG. 20B) the iliac crest, and (FIG. 20C) the tibia. FIG.20D is the quantitative RT-PCR analyses of endogenous osteogenic geneexpression in freshly harvested rat BGM from the indicated sources. FIG.20E illustrates a schematic of experimental design, where autologous BGMis transplanted into the SRC of rats. FIG. 20F exemplifiesrepresentative tissue sections of iliac crest BGM on post-transplant day7, stained to detect BrdU incorporation. Dotted lines indicatetrabecular bone chips included in the BGM. FIG. 20G, FIG. 20H, and FIG.20I are Runx2, Sox9, and PPARγ expressions, respectively. FIG. 20J showsrepresentative tissue sections of BGM stained with Aniline blue todetect osteoid matrix; asterisks indicate new bone matrix as opposed toold bone chips (yellow dotted line). The kidney surface is indicatedwith a dotted white line in this panel, and in FIG. 20G. FIG. 20K isSafranin 0/Fast green histology to detect proteoglycan-rich cartilage(red), and FIG. 20L is Gomori trichrome staining to detect adipocytes.Abbreviations: BrdU, bromodeoxyuridine; PPARγ, peroxisomeproliferator-activated protein gamma. Scale bars: 50 μm, asterisks:p<0.05.

FIG. 21A-21F illustrates bone graft material as Wnt responsive. FIG. 21Aindicates GFP^(+ve) cells in Axin2^(creERT2); R26^(mTmG) mice,visualized by immunostaining of the periosteum and FIG. 21B isendosteum. FIG. 21C is quantification of GFP^(+ve) cells/total cellswithin specified microscopic fields of view. FIG. 21D indicatesGFP^(+ve) cells in the BGM were visualized by fluorescence. FIG. 21E isquantitative absolute RT-PCR results for endogenous Axin2, Lef1, andGAPDH expression in BGM^(young) (green bars) and BGM^(aged) (grey bars).FIG. 21F illustrates western blot analyses for Wnt3a, total betacatenin, Axin2, and beta actin in in BGM^(young) (green bars) andBGM^(aged) (grey bars). Scale bars=50 μm. Asterisks: p<0.05.

FIG. 22A-22L shows osteogenic differentiation potential of BGM declineswith age. FIG. 22A indicates quantitative RT-PCR analyses for expressionof alkaline phosphatase, Osterix, and Osteocalcin in BGM^(young) (greenbars) and BGM^(aged) (grey bars). FIG. 22B is BGM harvested fromACTB-eGFP mice, transplanted into the SRC and visualized underbrightfield and FIG. 22C is the detection of the GFP signal in BGMutilizing fluorescent light. FIG. 22D is representative tissue sectionsstained with Aniline blue (inset) from BGM^(young) (N=5) and (FIG. 22E)BGM^(aged) (N=5). Dotted line indicates the kidney surface. FIG. 22Findicates histomorphometric analyses of Aniline blue^(+ve) pixels withinthe total area occupied by the BGM on post-transplant day 7. FIG. 22Gindicates representative tissue sections stained to detect ALP activityfrom BGM^(young) (N=5) and (FIG. 22H) BGM^(aged) (N=5). FIG. 22Iindicates quantification of ALP^(+ve) pixels within the total areaoccupied by the BGM on post-transplant day 7. FIG. 22J showsrepresentative tissue sections immunostained for GFP from BGM^(young)(N=5) and (FIG. 22K) BGM^(aged) (N=5). FIG. 22L shows quantification ofGFP^(+ve) pixels within the total area occupied by the BGM onpost-transplant day 7. Abbreviations: ALP, alkaline phosphatase; Oc,Osteocalcin. Scale bars: 100 μm. Asterisks: p<0.05; double asterisks:p<0.01.

FIG. 23A-23L shows osteogenic differentiation of BGM that requires anendogenous Wnt signal. FIG. 23A shows representative tissue sectionsstained for ALP activity in BGM treated with the murine IgG2α Fcfragment (Ad-Fc) or (FIG. 23B) adenovirus expressing the soluble Wntantagonist Dkk1 (Ad-Dkk1). FIG. 23C shows representative tissue sectionsimmunostained for PPARγ in BGM treated with Ad-Fc or (FIG. 23D) Ad-Dkk1.FIG. 23E illustrates representative tissue sections immunostained forDlk1 in BGM treated with Ad-Fc or (FIG. 23F) Ad-Dkk1. FIG. 23Gillustrates micro-CT reconstruction to detect bone formation in defectsites that received BGM treated with Ad-Fc or (FIG. 23H) Ad-Dkk1.Original defect is indicated with a dotted red circle. FIG. 23I showsnew bone volume (N=5) calculated from micro-CT data±SEM. FIG. 23Jindicates aniline blue staining on representative tissue sections fromdefect sites that received BGM treated with Ad-Fc or (FIG. 23K) Ad-Dkk1.FIG. 23L indicates quantification of new bone volume usinghistomorphometric analyses. FIG. 23I is PPAR-γ expression in BM graftstreated with Ad-Fc or (FIG. 23J) Ad-Dkk1. Single asterisk p<0.05. Scalebars: A-B, 200 μm, C-F, J-K, 50 μm, G-H, 2 mm.

FIG. 24A-24R illustrates human Wnt3a activation of BGM^(aged) andrestoration of its osteogenic differentiation potential. FIG. 24Aindicates BGMs from aged ACTB-eGFP mice, treated with L-PBS or L-WNT3A(0.15 μg/ml) for 1 h then either assayed by qRT-PCR for target geneexpression 24 h later, or immediately transplanted into the SRC for 7days. FIG. 24B illustrates fold change in Axin2 and Left expression inBGM^(aged) treated with either L-PBS (grey bars) or L-WNT3A (blue bars).FIG. 24C are western blot analysis of total beta catenin, Axin2, andbeta actin in BGM^(aged) treated with either L-PBS (grey bars) orL-WNT3A (blue bars). After harvesting BGM^(aged) from the SRC onpost-transplant day 4, representative tissue sections from (FIG. 24D)L-PBS (N=5) and (FIG. 24E) L-WNT3A were stained for BrdU incorporation(N=5). FIG. 24F indicates quantification of BrdU^(+ve) pixels within amicroscopic field of view centered in the middle of the bone grafts.FIG. 24G illustrates representative tissue sections from L-PBS (N=5) and(FIG. 24H) L-WNT3A treated (N=5) samples, stained for BrdU incorporationon post-transplant day 7. FIG. 24I illustrates quantification ofBrdU^(+ve) pixels as above. FIG. 24J indicates representative tissuesections from L-PBS (N=5) and (FIG. 24K) L-WNT3A treated (N=5) samples,immunostained for Dlk1 expression on post-transplant day 7. FIG. 24Lshows quantification of Dlk1^(+ve) pixels within the total area occupiedby the BGM on post-transplant day 7. FIG. 24M shows representativetissue sections from L-PBS (N=5) and (FIG. 24N) L-WNT3A treated (N=5)samples, immunostained for Oc expression on post-transplant day 7. FIG.24O indicates quantification of Oc^(+ve) pixels as described for Dlk1.FIG. 24P shows representative tissue sections stained with Aniline blueto detect osteoid matrix in L-PBS (N=5) and (FIG. 24Q) L-WNT3A treated(N=5) samples. FIG. 24R indicates histomorphometric quantification ofnew bone matrix. Abbreviations are described as elsewhere herein. Scalebars: 100 μm. Asterisks: p<0.05; double asterisks: p<0.01.

FIG. 25A-25J illustrates L-Wnt3a stimulation of BGM stem cells andimprovement of spinal fusion. FIG. 25A shows human MSC cultures weretreated with L-PBS or L-WNT3A at 37° C. for the time points indicatedand qRT-PCR for Axin2 expression was used to determine Wnt-response.FIG. 25B shows murine SSC were treated with L-PBS or L-WNT3A for 12 h at37° C. and Wnt response was assayed with qRT-PCR for Axin2 expression.FIG. 25C illustrates quantitative absolute RT-PCR analyses for Axin2 andLef1 expression in response to 1 h incubation at room temperature withL-PBS (dashed line) or L-WNT3A (0.15 μg/mL; blue line). Data isexpressed as a ratio of RNA copies/total RNA content over a 24 h period.FIG. 25D indicates rat spinous processes were exposed via minimalincisions and standardized volumes of autologous BGM from the iliaccrest were treated with L-PBS or L-WNT3A for 1 hr then (FIG. 25E)transplanted between the transverses processes of the L4 and L5vertebrae. FIG. 25F indicates at POD2 Micro-CT acquisitions wereperformed for graft (pink) treated with L-PBS and (FIG. 25G) L-WNT3A.FIG. 25H indicates at POD49 Micro-CT acquisitions were again performedto evaluate the bone growth of the transplants treated with L-PBS (gray)and (FIG. 25I) L-WNT3A (blue). FIG. 25J indicates transplant growth thatwas graphed for each of the treatment groups as fold volume, comparingeach graft size on POD2 to its size on POD49 (indicated by the colors asstated above). Abbreviations: L4, Lumbar #4, L5, Lumbar #5, AP, apicalprocess, SP, spinous process, TP, transverse process, POD,post-operation day.

FIG. 26 illustrates quantification of BGM volume. BGM harvested from thetibiae and femurs was divided into 5 aliquots of about ˜20 μL then DNAconcentration was evaluated as a means to quantify cellular density inthe heterogeneous mixture of cells, stroma, and bone fragments. The DNAconcentration in sample #1 was set as at 100, and the relative DNAconcentration of the other 4 aliquots were determined.

FIG. 27A-27L illustrates engraftment efficiency of BGM. L-Wnt3atreatment improves BGM engraftment efficiency. FIG. 27A illustrates GFPimmunostaining of BGM^(aged) and (FIG. 27B) BGM^(ACT) after 4 d in theSRC. FIG. 27C shows quantification of GFP^(+ve) pixels over total pixelsoccupied by the BGM on post-transplant day 4. FIG. 27D shows TUNELstaining of BGM^(aged) and (FIG. 27E) BGM^(ACT) after 4 d in the SRC.FIG. 27F shows quantification of TUNEL^(+ve) cells over total DAPI^(+ve)pixels within the total area occupied by the BGMs on post-transplantdays 4. FIG. 27G shows TUNEL staining of BGM^(aged) and (FIG. 27H)BGM^(ACT) after 7 d in the SRC. FIG. 27I shows quantification ofTUNEL^(+ve) cells over total DAPI^(+ve) pixels within the total areaoccupied by the BGMs on post-transplant day 7. FIG. 27J illustratestissue sections stained for tartrate resistant acid phosphatase (TRAP)activity to detect osteoclasts in BGM^(aged) and (FIG. 27K) BGM^(ACT)after 7 d in the SRC. FIG. 27L shows quantification of TRAP^(+ve) pixelswithin the total area occupied by the BGMs on post-transplant days 7.Scale Bars=100 μm, ** indicates p<0.01.

DETAILED DESCRIPTION OF THE INVENTION

Disclosed herein are compositions, methods, processes, and kits for usein generating a liposomal Wnt polypeptide. Also disclosed herein arecompositions, methods, processes, and kits for generating bone graftmaterials. In some embodiments, a composition comprises a functionallyactive mammalian Wnt polypeptide and an aqueous solution comprisingliposomes, wherein the phospholipids comprising the liposomes have aphase transition temperature from about 10° C. to about 25° C. In someembodiments, a composition comprises a functionally active mammalian Wntpolypeptide and an aqueous solution comprising liposomes, wherein thephospholipids comprising the liposomes have a tail carbon length ofbetween about 12 carbons and about 14 carbons. In some embodiments, acomposition also comprises a functionally active Wnt3a polypeptide andan aqueous solution comprising liposomes, wherein the functionallyactive Wnt3a polypeptide comprises a lipid modification at an amino acidposition corresponding to amino acid residue 209 as set forth in SEQ IDNO:1.

In some embodiments, described herein is a method of preparing aliposomal Wnt polypeptide, which comprises the steps of contacting asample comprising Wnt polypeptides to an aqueous solution of liposomes,wherein the phospholipids comprising the liposomes have a phasetransition temperature from about 10° C. to about 25° C. In someembodiments, described herein is a method of preparing a liposomal Wntpolypeptide, which comprises the steps of contacting a sample comprisingWnt polypeptides to an aqueous solution of liposomes, wherein thephospholipids comprising the liposome have a tail carbon length ofbetween about 12 carbons and about 14 carbons. In some embodiments, alsodescribed herein is a method of preparing a liposomal Wnt3a polypeptide,which comprises the steps of (a) harvesting Wnt3a polypeptides from aconditioned media comprising Chinese hamster ovary (CHO) cells; (b)introducing the Wnt3a polypeptides to an ion-exchange column immobilizedwith a sulfonated polyaromatic compound; (c) eluting the Wnt3apolypeptides from the ion-exchange column utilizing a step gradient; and(d) contacting the Wnt3a polypeptides from step (c) with an aqueoussolution of liposomes.

In some embodiments, described herein is a composition of isolatedenhanced mammalian bone marrow cells wherein the cells have an enhancedexpression level in one or more of the biomarkers selected from thegroup consisting of: Runx2, Osterix, Osteocalcin, Osteopontin, alkalinephosphatase, collagen type I, Axin2, Lef1, and Tcf4, after treatmentwith a liposomal Wnt polypeptide. In some embodiments, also describedherein is a composition of mammalian bone marrow cells obtained from ahuman subject at or older than 35 years of age, wherein the mammalianbone marrow cells express an enhanced expression level in one or more ofthe biomarkers selected from the group consisting of: Runx2, Osterix,Osteocalcin, Osteopontin, alkaline phosphatase, collagen type I, Axin2,Lef1, and Tcf4, after treatment with a liposomal Wnt polypeptide.

In some embodiments, described herein is a mammalian bone marrowcomposition produced by a process which comprises the step of contactingisolated mammalian bone marrow cells ex-vivo with a liposomal Wntpolypeptide, wherein the contacting time is between about 30 minutes andabout 4 hours.

In some embodiments, described herein is a method of treating a bonedefect in a subject, which comprises the steps of (a) contacting asample comprising mammalian bone marrow cells ex-vivo with a liposomalWnt polypeptide; (b) washing the sample to remove free liposomal Wntpolypeptide; and (c) transplanting the liposomal Wnt polypeptide treatedbone graft materials into a site of bone defect. Also described hereinis a method of treating a bone defect in a subject, which comprises thesteps of (a) contacting a sample comprising mammalian bone marrow cellsex-vivo with a liposomal Wnt3a polypeptide; (b) washing the sample toremove free liposomal Wnt3a polypeptide; and (c) transplanting theliposomal Wnt3a polypeptide treated bone graft materials into a site ofbone defect.

In some embodiments, described herein is a method of generating bonegraft materials, which comprises the steps of (a) contacting a samplecomprising mammalian bone marrow cells ex-vivo with a liposomal Wntpolypeptide; (b) washing the sample to remove free liposomal Wntpolypeptide; and (c) transplanting the liposomal Wnt polypeptide treatedbone graft materials into a site of bone defect. Also described hereinis a method of generating bone graft materials, which comprises thesteps of (a) contacting a sample comprising mammalian bone marrow cellsex-vivo with a liposomal Wnt3a polypeptide; (b) washing the sample toremove free liposomal Wnt3a polypeptide; and (c) transplanting theliposomal Wnt3a polypeptide treated bone graft materials into a site ofbone defect.

In some aspects, described herein is a composition which comprises atleast about 60%, or at least about 70% or at least about 80%, or atleast about 85%, or at least about 90%, or at least about 95% offunctionally active Wnt3a polypeptide of SEQ ID NO: 1 lipid modified atSer209, or at a corresponding conserved Ser of a non-human mammalianWnt3a polypeptide in the absence of lipid modification at Cys77; or at acorresponding conserved Cys of a non-human mammalian Wnt3a polypeptide.In some embodiments, described herein is a composition which comprisesat least about 60%, or at least about 70% or at least about 80%, or atleast about 85%, or at least about 90%, or at least about 95% offunctionally active human Wnt polypeptide other than Wnt3A, which islipid modified at a conserved Ser corresponding to Wnt3A ser209 in theabsence of lipid modification at a corresponding conserved Cys to Wnt3ACys77.

In some aspects, the Wnt polypeptide is Wnt3a or a functionally activevariant therof. In other aspects, the Wnt polypeptide is human Wnt3a ora functionally active variant thereof. In yet another aspect, the Wntpolypeptide is Wnt3a of SEQ ID NO: 1 or a functionally active varianttherof. In a further aspect, the lipid modification is palmitoylation.In other aspects, the Wnt polypeptide is a human Wnt protein other thanWnt3A, such as a Wnt1, Wnt2, Wnt2b (or Wnt13), Wnt3, Wnt4, Wnt5a, Wnt5b,Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a (Wnt14, or Wnt14b), Wnt9b(Wnt14b, or Wnt15), Wnt10a, Wnt10b (or Wnt12), Wnt11, Wnt-16a, Wnt-16bpolypeptide or a functionally active variant therof. In someembodiments, the polypeptide is Wnt5A or Wnt10b or a functionally activevariant therof. In some such embodiments, the Wnt polypeptide isformulated into a liposome, e.g. a liposome comprising DMPC and/orcholesterol.

In another aspect, described herein is a method for the purification ofa Wnt polypeptide from mammalian cell culture, including withoutlimitation, recombinant Chinese Hamster Ovary (CHO) cells, whichcomprises subjecting a culture medium of said cells that contains theWnt polypeptide to purification utilizing a separation step such as forexample on a chromatographic column (e.g. Blue Sepharose ion-exchangecolumn) in the absence of alternative purification steps such as a gelfiltration purification step. In some embodiments, the purification isperformed also in the absence of a purification step of a heparinsulfate column. In another embodiment, the cells are adherent cells andthe culture medium further comprises serum, such as fetal bovine serum(FBS). In further embodiments, purification on columns such as the BlueSepharose ion-exchange column is performed using a salt gradient of 150mM to 1.5 M, where the salt can, for example be sodium or potassiumchloride. In some instances, the purification scheme is followed by afurther purification step such as for example using pre-fabricatedliposomes which can comprise lipids such as DPMC and/or cholesterol, forexample at a 90:10 mol:mol ratio. In some instances, the use ofpre-fabricated liposomes takes advantage of the hydrophobicity of Wntpolypeptides and eliminates the need for further purification steps,such as gel filtration and/or heparin sulfate column purification steps.

When the cells are suspension cells, culturing can be performed underserum-free conditions. In some embodiments, the Wnt polypeptide can beWnt3a, such as human Wnt3a, e.g. human Wnt3a of SEQ ID NO: 1 or afunctionally active variant therof; or the Wnt polypeptide can be ahuman Wnt polypeptide other than Wnt3A, e.g. Wnt1, Wnt2, Wnt2b (orWnt13), Wnt3, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b,Wnt9a (Wnt14, or Wnt14b), Wnt9b (Wnt14b, or Wnt15), Wnt10a, Wnt10b (orWnt12), Wnt11, Wnt-16a, Wnt-16b polypeptide or a functionally activevariant therof. In some embodiments, the polypeptide is Wnt5A or Wnt10bor a functionally active variant therof. In a further aspect, theinvention concerns a Wnt composition purified by the foregoing method.In one embodiment, the Wnt composition comprises a Wnt polypeptide thatis Wnt3a or a functionally active variant therof. In another embodiment,the Wnt composition comprises a Wnt polypeptide that is human Wnt3a or afunctionally active variant therof. In yet another embodiment, the Wntcomposition comprises a Wnt polypeptide that is human Wnt3a of SEQ IDNO: 1 or a functionally active variant therof.

In some aspects, described herein also includes kits for generating bonegraft materials which comprise the liposomal Wnt polypeptide, theliposomal Wnt3a polypeptide, or the isolated enhanced mammalian bonemarrow cell compositions.

A. Definitions

Before the present methods are described, it is to be understood thatthis invention is not limited to particular methods described, as suchcan, of course, vary. It is also to be understood that the terminologyused herein is for the purpose of describing particular embodimentsonly, and is not intended to be limiting, since the scope of the presentinvention will be limited only by the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range is encompassed within the invention. The upper and lowerlimits of these smaller ranges can independently be included in thesmaller ranges encompassed within the invention, subject to anyspecifically excluded limit in the stated range. Also used herein,ranges and amounts can be expressed as “about” a particular value orrange. About also includes the exact amount. Hence “about 5 μL” means“about 5 μL” and also “5 μL.” Generally, the term “about” includes anamount that would be expected to be within ±10% of the amount.

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Singleton et al, Dictionary ofMicrobiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York,N.Y. 1994), provides one skilled in the art with a general guide to manyof the terms used in the present application.

Wnt polypeptides form a family of highly conserved secreted signalingmolecules that regulate cell-to-cell interactions during embryogenesis.The terms “Wnts” or “Wnt gene product” or “Wnt polypeptide” when usedherein encompasses native sequence Wnt polypeptides, Wnt polypeptidefragments, chimeric Wnt polypeptides or functionally active variants ofthe foregoing. The terms “WNT” and “Wnt” are used interchangeablyherein. Similarly, the terms“WNT3A”, “WNT3a”, “Wnt3A”, or “Wnt3a” areused interchangeably herein.

A “native sequence” polypeptide is one that has the same amino acidsequence as a Wnt polypeptide derived from nature. Such native sequencepolypeptides can be isolated from cells producing endogenous Wnt proteinor can be produced by recombinant or synthetic means. Thus, a nativesequence polypeptide can have the amino acid sequence of, e.g. naturallyoccurring human polypeptide, murine polypeptide, or polypeptide from anyother mammalian species, or from non-mammalian species, e.g. Drosophila,C. elegans, or the like. In some instances, the “native sequence”polypeptide also includes the N-terminal methionine. In some instances,it does not include the N-terminal methionine.

The term “native sequence Wnt polypeptide” includes, without limitation,mammalian Wnt polypeptides, such as human or murine Wnt polypeptides.Human Wnt polypeptides include Wnt1, Wnt2, Wnt2b (or Wnt13), Wnt3,Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a(Wnt14, or Wnt14b), Wnt9b (Wnt14b, or Wnt15), Wnt10a, Wnt10b (or Wnt12),Wnt11, Wnt-16a, or Wnt-16b polypeptide. Wnt1 can be referred by theGenbank references NP005421.1 and AAH74799.1. Wnt2 can be referred bythe Genbank references NP003382.1 and AAH78170.1 In general, Wnt2 can beexpressed in the brain, thalamus, in both fetal and adult lungs, or inthe placenta. Wnt2B has two isoforms and their Genbank reference Nos.are NP004176.2 and NP078613.1, respectively. Isoform 1 can be expressedin adult heart, brain, placenta, lung, prostate, testis, ovary, smallintestine and/or colon. In the adult brain, it is mainly found in thecaudate nucleus, subthalamic nucleus and thalamus. In some instances, itis also detected in fetal brain, lung and kidney. Isoform 2 can beexpressed in fetal brain, fetal lung, fetal kidney, caudate nucleus,testis and/or cancer cell lines.

Wnt3 and Wnt3a play distinct roles in cell-cell signaling duringmorphogenesis of the developing neural tube. Wnt3 has the Genbankreference AB060284.1 (see also GenBank Nos. BAB61052.1 and AA103924.1.Wnt3a has the amino acid sequence as set forth in SEQ ID NO:1 and thenucleic acid sequence as set forth in SEQ ID NO:2. The native humanWnt3a amino acid and nucleotide sequences are also disclosed in Genbankreference NM_033131, and the protein sequence at Genbank referenceNP_149122. The Wnt3a protein precursor has the Genbank referenceNP_149122.1. The native Wnt3a is 352 amino acid residues in length. Itcontains a signal peptide which is from amino acid residues 1-18. Themature protein contains the amino acid residues 19-352. Reference to,for example, Cys77 or Ser209 of Wnt3A, as used herein, is made relativeto the full-length sequence, as shown in SEQ ID NO:1. In some instances,the term “native human Wnt3a” polypeptide refers to the native humanWnt3a polypeptide of SEQ ID NO:1, with or without its N-terminalmethionine (Met), and with or without the native signal sequence.

Wnt4 has the Genbank references NP110388.2 and BAC23080.1. Wnt 5a hasthe Genbank references NP003383.1, and NP003383.2.Wnt5b has the Genbankreferences BAB62039.1 and AAG38659. Wnt 6 has the Genbank referencesNP006513.1 and BAB55603.1. Wnt 7a has the Genbank references NP004616.2and BAA82509.1. It can be expressed in the placenta, kidney, testis,uterus, fetal lung, fetal brain, or adult brain. Wnt 7b has the Genbankreferences NP4 78679.1 and BAB68399.1. It can be expressed in fetalbrain, lung and/or kidney, or in adult brain, lung and/or prostate. Wnt8A has at least two alternative transcripts, Genbank references NP114139.1 and NP490645.1. Wnt 8B can be expressed in the forebrain. Ithas the Genbank reference NP003384.1. Wnt 10A has the Genbank referencesAAG45153 and NP079492.2. Wnt 10B is detected in most adult tissues, withhighest levels in the heart and skeletal muscles. It has the Genbankreference NP003385.2. Wnt 11 can be expressed in fetal lung, kidney,adult heart, liver, skeletal muscle, and pancreas. It has the Genbankreference NP004617 0.2. Wnt 14 has the Genbank reference NP003386.1. Wnt15 can be expressed in fetal kidney or adult kidney. It can also beexpressed in the brain. It has the Genbank reference NP003387.1. Wnt 16has two isoforms, Wnt-16a and Wnt-16b, produced by alternative splicing.Isoform Wnt-16a can be expressed in the pancreas. Isoform Wnt-16b can beexpressed in peripheral lymphoid organs such as spleen, appendix, andlymph nodes, or in the kidney. However, it cannot be expressed in bonemarrow. The Genbank references are NP476509.1 and NP057171.2,respectively, for Wnt16a and Wnt16b. All GenBank, SwissProt and otherdatabase sequences listed are expressly incorporated by referenceherein.

A “variant” polypeptide means a functionally active polypeptide asdefined below having less than 100% sequence identity with a nativesequence polypeptide. Such variants include polypeptides longer orshorter polypeptides, for example wherein one or more amino acidresidues are added at the N- or C terminus of, or within, the nativesequence; from about one to 300 amino acid residues are deleted. Variantpolypeptides include polypeptides with one or more amino acidsubstitutions in comparison to the native polypeptide sequence, orderivatives of a polypeptide sequence, wherein an amino acid residue hasbeen covalently modified so that the resulting product has anon-naturally occurring amino acid.

A “functionally active” Wnt polypeptide (e.g. Wnt3a polypeptide) retainsthe effector functions that are directly or indirectly caused orperformed by native sequence Wnt polypeptides. Effector functions ofnative sequence Wnt polypeptides include stabilization of β-catenin,stimulation of stem cell self-renewal, C57MG transformation andinduction of target genes in Xenopus animal cap assays, as well astarget gene expression in human teratocarcinoma cells. The purified Wntcompositions find use in a variety of therapeutic methods, including themaintenance and growth of stem cells, tissue regeneration, and the like.

In some instances, a functionally active Wnt variant will have an aminoacid sequence that has at least about 30%, at least about 40%, at leastabout 50%, at least about 60%, at least about 70%, at least about 80%,at least about 90%, at least about 95%, or at least about 99% amino acidsequence identity with a native sequence Wnt polypeptide. In oneembodiment the native sequence Wnt polypeptide is a mammalian (e.g.,human) Wnt polypeptide such as Wnt3a.

The term “amino acid” refers to a molecule containing both an aminogroup and a carboxyl group. Suitable amino acids include, withoutlimitation, both the D- and L-isomers of the naturally-occurring aminoacids, as well as non-naturally occurring amino acids prepared byorganic synthesis or other metabolic routes. The term amino acid, asused herein, includes, without limitation, α-amino acids, natural aminoacids, non-natural amino acids, and amino acid analogs.

The term “α-amino acid” refers to a molecule containing both an aminogroup and a carboxyl group bound to a carbon which is designated theα-carbon.

The term “β-amino acid” refers to a molecule containing both an aminogroup and a carboxyl group in a β configuration.

The term “naturally occurring amino acid” refers to any one of thetwenty amino acids commonly found in peptides synthesized in nature, andknown by the one letter abbreviations A, R, N, C, D, Q, E, G, H, I, L,K, M, F, P, S, T, W, Y and V.

The following table shows a summary of the properties of natural aminoacids:

3- 1- Side- Side-chain Letter Letter chain charge Hydropathy Amino AcidCode Code Polarity (pH 7.4) Index Alanine Ala A nonpolar neutral 1.8Arginine Arg R polar positive −4.5 Asparagine Asn N polar neutral −3.5Aspartic acid Asp D polar negative −3.5 Cysteine Cys C polar neutral 2.5Glutamic acid Glu E polar negative −3.5 Glutamine Gln Q polar neutral−3.5 Glycine Gly G nonpolar neutral −0.4 Histidine His H polar positive−3.2 (10%) neutral (90%) Isoleucine Ile I nonpolar neutral 4.5 LeucineLeu L nonpolar neutral 3.8 Lysine Lys K polar positive −3.9 MethionineMet M nonpolar neutral 1.9 Phenylalanine Phe F nonpolar neutral 2.8Proline Pro P nonpolar neutral −1.6 Serine Ser S polar neutral −0.8Threonine Thr T polar neutral −0.7 Tryptophan Trp W nonpolar neutral−0.9 Tyrosine Tyr Y polar neutral −1.3 Valine Val V nonpolar neutral 4.2

“Hydrophobic amino acids” include small hydrophobic amino acids andlarge hydrophobic amino acids. “Small hydrophobic amino acid” areglycine, alanine, proline, and analogs thereof “Large hydrophobic aminoacids” are valine, leucine, isoleucine, phenylalanine, methionine,tryptophan, and analogs thereof. “Polar amino acids” are serine,threonine, asparagine, glutamine, cysteine, tyrosine, and analogsthereof “Charged amino acids” are lysine, arginine, histidine,aspartate, glutamate, and analogs thereof.

The term “amino acid analog” refers to a molecule which is structurallysimilar to an amino acid and which can be substituted for an amino acidin the formation of a peptidomimetic macrocycle Amino acid analogsinclude, without limitation, β-amino acids and amino acids where theamino or carboxy group is substituted by a similarly reactive group(e.g., substitution of the primary amine with a secondary or tertiaryamine, or substitution of the carboxy group with an ester).

The term “non-natural amino acid” refers to an amino acid which is notone of the twenty amino acids commonly found in peptides synthesized innature, and known by the one letter abbreviations A, R, N, C, D, Q, E,G, H, I, L, K, M, F, P, S, T, W, Y and V. Non-natural amino acids oramino acid analogs include, without limitation, structures according tothe following:

Amino acid analogs include β-amino acid analogs. Examples of β-aminoacid analogs include, but are not limited to, the following: cyclicβ-amino acid analogs; β-alanine; (R)-β-phenylalanine;(R)-1,2,3,4-tetrahydro-isoquinoline-3-acetic acid;(R)-3-amino-4-(1-naphthyl)-butyric acid;(R)-3-amino-4-(2,4-dichlorophenyl)butyric acid;(R)-3-amino-4-(2-chlorophenyl)-butyric acid;(R)-3-amino-4-(2-cyanophenyl)-butyric acid;(R)-3-amino-4-(2-fluorophenyl)-butyric acid;(R)-3-amino-4-(2-furyl)-butyric acid;(R)-3-amino-4-(2-methylphenyl)-butyric acid;(R)-3-amino-4-(2-naphthyl)-butyric acid;(R)-3-amino-4-(2-thienyl)-butyric acid;(R)-3-amino-4-(2-trifluoromethylphenyl)-butyric acid;(R)-3-amino-4-(3,4-dichlorophenyl)butyric acid;(R)-3-amino-4-(3,4-difluorophenyl)butyric acid;(R)-3-amino-4-(3-benzothienyl)-butyric acid;(R)-3-amino-4-(3-chlorophenyl)-butyric acid;(R)-3-amino-4-(3-cyanophenyl)-butyric acid;(R)-3-amino-4-(3-fluorophenyl)-butyric acid;(R)-3-amino-4-(3-methylphenyl)-butyric acid;(R)-3-amino-4-(3-pyridyl)-butyric acid;(R)-3-amino-4-(3-thienyl)-butyric acid;(R)-3-amino-4-(3-trifluoromethylphenyl)-butyric acid;(R)-3-amino-4-(4-bromophenyl)-butyric acid;(R)-3-amino-4-(4-chlorophenyl)-butyric acid;(R)-3-amino-4-(4-cyanophenyl)-butyric acid;(R)-3-amino-4-(4-fluorophenyl)-butyric acid;(R)-3-amino-4-(4-iodophenyl)-butyric acid;(R)-3-amino-4-(4-methylphenyl)-butyric acid;(R)-3-amino-4-(4-nitrophenyl)-butyric acid;(R)-3-amino-4-(4-pyridyl)-butyric acid;(R)-3-amino-4-(4-trifluoromethylphenyl)-butyric acid;(R)-3-amino-4-pentafluoro-phenylbutyric acid; (R)-3-amino-5-hexenoicacid; (R)-3-amino-5-hexynoic acid; (R)-3-amino-5-phenylpentanoic acid;(R)-3-amino-6-phenyl-5-hexenoic acid;(S)-1,2,3,4-tetrahydro-isoquinoline-3-acetic acid;(S)-3-amino-4-(1-naphthyl)-butyric acid;(S)-3-amino-4-(2,4-dichlorophenyl)butyric acid;(S)-3-amino-4-(2-chlorophenyl)-butyric acid;(S)-3-amino-4-(2-cyanophenyl)-butyric acid;(S)-3-amino-4-(2-fluorophenyl)-butyric acid;(S)-3-amino-4-(2-furyl)-butyric acid;(S)-3-amino-4-(2-methylphenyl)-butyric acid;(S)-3-amino-4-(2-naphthyl)-butyric acid;(S)-3-amino-4-(2-thienyl)-butyric acid;(S)-3-amino-4-(2-trifluoromethylphenyl)-butyric acid;(S)-3-amino-4-(3,4-dichlorophenyl)butyric acid;(S)-3-amino-4-(3,4-difluorophenyl)butyric acid;(S)-3-amino-4-(3-benzothienyl)-butyric acid;(S)-3-amino-4-(3-chlorophenyl)-butyric acid;(S)-3-amino-4-(3-cyanophenyl)-butyric acid;(S)-3-amino-4-(3-fluorophenyl)-butyric acid;(S)-3-amino-4-(3-methylphenyl)-butyric acid;(S)-3-amino-4-(3-pyridyl)-butyric acid;(S)-3-amino-4-(3-thienyl)-butyric acid;(S)-3-amino-4-(3-trifluoromethylphenyl)-butyric acid;(S)-3-amino-4-(4-bromophenyl)-butyric acid;(S)-3-amino-4-(4-chlorophenyl) butyric acid;(S)-3-amino-4-(4-cyanophenyl)-butyric acid;(S)-3-amino-4-(4-fluorophenyl) butyric acid;(S)-3-amino-4-(4-iodophenyl)-butyric acid;(S)-3-amino-4-(4-methylphenyl)-butyric acid;(S)-3-amino-4-(4-nitrophenyl)-butyric acid;(S)-3-amino-4-(4-pyridyl)-butyric acid;(S)-3-amino-4-(4-trifluoromethylphenyl)-butyric acid;(S)-3-amino-4-pentafluoro-phenylbutyric acid; (S)-3-amino-5-hexenoicacid; (S)-3-amino-5-hexynoic acid; (S)-3-amino-5-phenylpentanoic acid;(S)-3-amino-6-phenyl-5-hexenoic acid;1,2,5,6-tetrahydropyridine-3-carboxylic acid;1,2,5,6-tetrahydropyridine-4-carboxylic acid;3-amino-3-(2-chlorophenyl)-propionic acid;3-amino-3-(2-thienyl)-propionic acid;3-amino-3-(3-bromophenyl)-propionic acid;3-amino-3-(4-chlorophenyl)-propionic acid;3-amino-3-(4-methoxyphenyl)-propionic acid;3-amino-4,4,4-trifluoro-butyric acid; 3-aminoadipic acid;D-β-phenylalanine; β-leucine; L-β-homoalanine; L-β-homoaspartic acidγ-benzyl ester; L-β-homoglutamic acid δ-benzyl ester;L-β-homoisoleucine; L-β-homoleucine; L-β-homomethionine;L-β-homophenylalanine; L-β-homoproline; L-β-homotryptophan;L-β-homovaline; L-Nω-benzyloxycarbonyl-β-homolysine;Nω-L-β-homoarginine; O-benzyl-L-β-homohydroxproline;O-benzyl-L-β-homoserine; O-benzyl-L-β-homothreonine;O-benzyl-L-β-homotyrosine; γ-trityl-L-β-homoasparagine;(R)-β-phenylalanine; L-β-homoaspartic acid γ-t-butyl ester;L-β-homoglutamic acid δ-t-butyl ester; L-Nω-β-homolysine;Nδ-trityl-L-β-homoglutamine;Nω-2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl-L-β-homoarginine;O-t-butyl-L-β-homohydroxy-proline; O-t-butyl-L-β-homoserine;O-t-butyl-L-β-homothreonine; O-t-butyl-L-β-homotyrosine;2-aminocyclopentane carboxylic acid; and 2-aminocyclohexane carboxylicacid.

Amino acid analogs include analogs of alanine, valine, glycine orleucine. Examples of amino acid analogs of alanine, valine, glycine, andleucine include, but are not limited to, the following:α-methoxyglycine; α-allyl-L-alanine; α-aminoisobutyric acid;α-methyl-leucine; β-(1-naphthyl)-D-alanine; β-(1-naphthyl)-L-alanine;β-(2-naphthyl)-D-alanine; β-(2-naphthyl)-L-alanine;β-(2-pyridyl)-D-alanine; β-(2-pyridyl)-L-alanine;β-(2-thienyl)-D-alanine; β-(2-thienyl)-L-alanine;β-(3-benzothienyl)-D-alanine; β-(3-benzothienyl)-L-alanine;β-(3-pyridyl)-D-alanine; β-(3-pyridyl)-L-alanine;β-(4-pyridyl)-D-alanine; β-(4-pyridyl)-L-alanine; β-chloro-L-alanine;β-cyano-L-alanin; β-cyclohexyl-D-alanine; β-cyclohexyl-L-alanine;β-cyclopenten-1-yl-alanine; β-cyclopentyl-alanine;β-cyclopropyl-L-Ala-OH.dicyclohexylammonium salt; β-t-butyl-D-alanine;β-t-butyl-L-alanine; γ-aminobutyric acid; L-α,β-diaminopropionic acid;2,4-dinitro-phenylglycine; 2,5-dihydro-D-phenylglycine;2-amino-4,4,4-trifluorobutyric acid; 2-fluoro-phenylglycine;3-amino-4,4,4-trifluoro-butyric acid; 3-fluoro-valine;4,4,4-trifluoro-valine; 4,5-dehydro-L-leu-OH.dicyclohexylammonium salt;4-fluoro-D-phenylglycine; 4-fluoro-L-phenylglycine;4-hydroxy-D-phenylglycine; 5,5,5-trifluoro-leucine; 6-aminohexanoicacid; cyclopentyl-D-Gly-OH.dicyclohexylammonium salt;cyclopentyl-Gly-OH.dicyclohexylammonium salt; D-α,β-diaminopropionicacid; D-α-aminobutyric acid; D-α-t-butylglycine; D-(2-thienyl)glycine;D-(3-thienyl)glycine; D-2-aminocaproic acid; D-2-indanylglycine;D-allylglycine-dicyclohexylammonium salt; D-cyclohexylglycine;D-norvaline; D-phenylglycine; β-aminobutyric acid; β-aminoisobutyricacid; (2-bromophenyl)glycine; (2-methoxyphenyl)glycine;(2-methylphenyl)glycine; (2-thiazoyl)glycine; (2-thienyl)glycine;2-amino-3-(dimethylamino)-propionic acid; L-α,β-diaminopropionic acid;L-α-aminobutyric acid; L-α-t-butylglycine; L-(3-thienyl)glycine;L-2-amino-3-(dimethylamino)-propionic acid; L-2-aminocaproic aciddicyclohexyl-ammonium salt; L-2-indanylglycine;L-allylglycine.dicyclohexyl ammonium salt; L-cyclohexylglycine;L-phenylglycine; L-propargylglycine; L-norvaline;N-α-aminomethyl-L-alanine; D-α,γ-diaminobutyric acid;L-α,γ-diaminobutyric acid; β-cyclopropyl-L-alanine;(N-β-(2,4-dinitrophenyl))-L-α,μ-diaminopropionic acid;(N-β-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl)-D-α,μ-diaminopropionicacid;(N-β-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl)-L-α,μ-diaminopropionicacid; (N-β-4-methyltrityl)-L-α,β-diaminopropionic acid;(N-β-allyloxycarbonyl)-L-α,β-diaminopropionic acid;(N-γ-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl)-D-α,γ-diaminobutyricacid;(N-γ-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl)-L-α,γ-diaminobutyricacid; (N-γ-4-methyltrityl)-D-α,γ-diaminobutyric acid;(N-γ-4-methyltrityl)-L-α,γ-diaminobutyric acid;(N-γ-allyloxycarbonyl)-L-α,γ-diaminobutyric acid; D-α,γ-diaminobutyricacid; 4,5-dehydro-L-leucine; cyclopentyl-D-Gly-OH; cyclopentyl-Gly-OH;D-allylglycine; D-homocyclohexylalanine; L-1-pyrenylalanine;L-2-aminocaproic acid; L-allylglycine; L-homocyclohexylalanine; andN-(2-hydroxy-4-methoxy-Bzl)-Gly-OH.

Amino acid analogs include analogs of arginine or lysine. Examples ofamino acid analogs of arginine and lysine include, but are not limitedto, the following: citrulline; L-2-amino-3-guanidinopropionic acid;L-2-amino-3-ureidopropionic acid; L-citrulline; Lys(Me)₂-OH; Lys(N₃)—OH;Nδ-benzyloxycarbonyl-L-ornithine; Nω-nitro-D-arginine;Nω-nitro-L-arginine; α-methyl-ornithine; 2,6-diaminoheptanedioic acid;L-ornithine;(Nδ-1-(4,4-dimethyl-2,6-dioxo-cyclohex-1-ylidene)ethyl)-D-ornithine;(Nδ-1-(4,4-dimethyl-2,6-dioxo-cyclohex-1-ylidene)ethyl)-L-ornithine;(Nδ-4-methyltrityl)-D-ornithine; (Nδ-4-methyltrityl)-L-ornithine;D-ornithine; L-ornithine; Arg(Me)(Pbf)-OH; Arg(Me)₂-OH (asymmetrical);Arg(Me)2-OH (symmetrical); Lys(ivDde)-OH; Lys(Me)2-OH.HCl; Lys(Me3)-OHchloride; Nω-nitro-D-arginine; and Nω-nitro-L-arginine.

Amino acid analogs include analogs of aspartic or glutamic acids.Examples of amino acid analogs of aspartic and glutamic acids include,but are not limited to, the following: α-methyl-D-aspartic acid;α-methyl-glutamic acid; α-methyl-L-aspartic acid; γ-methylene-glutamicacid; (N-γ-ethyl)-L-glutamine; [N-α-(4-aminobenzoyl)]-L-glutamic acid;2,6-diaminopimelic acid; L-α-aminosuberic acid; D-2-aminoadipic acid;D-α-aminosuberic acid; α-aminopimelic acid; iminodiacetic acid;L-2-aminoadipic acid; threo-β-methyl-aspartic acid; γ-carboxy-D-glutamicacid γ,γ-di-t-butyl ester; γ-carboxy-L-glutamic acid γ,γ-di-t-butylester; Glu(OAII)-OH; L-Asu(OtBu)—OH; and pyroglutamic acid.

Amino acid analogs include analogs of cysteine and methionine. Examplesof amino acid analogs of cysteine and methionine include, but are notlimited to, Cys(farnesyl)-OH, Cys(farnesyl)-OMe, α-methyl-methionine,Cys(2-hydroxyethyl)-OH, Cys(3-aminopropyl)-OH,2-amino-4-(ethylthio)butyric acid, buthionine, buthioninesulfoximine,ethionine, methionine methylsulfonium chloride, selenomethionine,cysteic acid, [2-(4-pyridyl)ethyl]-DL-penicillamine,[2-(4-pyridyl)ethyl]-L-cysteine, 4-methoxybenzyl-D-penicillamine,4-methoxybenzyl-L-penicillamine, 4-methylbenzyl-D-penicillamine,4-methylbenzyl-L-penicillamine, benzyl-D-cysteine, benzyl-L-cysteine,benzyl-DL-homocysteine, carbamoyl-L-cysteine, carboxyethyl-L-cysteine,carboxymethyl-L-cysteine, diphenylmethyl-L-cysteine, ethyl-L-cysteine,methyl-L-cysteine, t-butyl-D-cysteine, trityl-L-homocysteine,trityl-D-penicillamine, cystathionine, homocystine, L-homocystine,(2-aminoethyl)-L-cysteine, seleno-L-cystine, cystathionine,Cys(StBu)—OH, and acetamidomethyl-D-penicillamine.

Amino acid analogs include analogs of phenylalanine and tyrosine.Examples of amino acid analogs of phenylalanine and tyrosine includeβ-methyl-phenylalanine, β-hydroxyphenylalanine,α-methyl-3-methoxy-DL-phenylalanine, α-methyl-D-phenylalanine,α-methyl-L-phenylalanine, 1,2,3,4-tetrahydroisoquinoline-3-carboxylicacid, 2,4-dichloro-phenylalanine, 2-(trifluoromethyl)-D-phenylalanine,2-(trifluoromethyl)-L-phenylalanine, 2-bromo-D-phenylalanine,2-bromo-L-phenylalanine, 2-chloro-D-phenylalanine,2-chloro-L-phenylalanine, 2-cyano-D-phenylalanine,2-cyano-L-phenylalanine, 2-fluoro-D-phenylalanine,2-fluoro-L-phenylalanine, 2-methyl-D-phenylalanine,2-methyl-L-phenylalanine, 2-nitro-D-phenylalanine,2-nitro-L-phenylalanine, 2;4;5-trihydroxy-phenylalanine,3,4,5-trifluoro-D-phenylalanine, 3,4,5-trifluoro-L-phenylalanine,3,4-dichloro-D-phenylalanine, 3,4-dichloro-L-phenylalanine,3,4-difluoro-D-phenylalanine, 3,4-difluoro-L-phenylalanine,3,4-dihydroxy-L-phenylalanine, 3,4-dimethoxy-L-phenylalanine,3,5,3′-triiodo-L-thyronine, 3,5-diiodo-D-tyrosine,3,5-diiodo-L-tyrosine, 3,5-diiodo-L-thyronine,3-(trifluoromethyl)-D-phenylalanine,3-(trifluoromethyl)-L-phenylalanine, 3-amino-L-tyrosine,3-bromo-D-phenylalanine, 3-bromo-L-phenylalanine,3-chloro-D-phenylalanine, 3-chloro-L-phenylalanine, 3-chloro-L-tyrosine,3-cyano-D-phenylalanine, 3-cyano-L-phenylalanine,3-fluoro-D-phenylalanine, 3-fluoro-L-phenylalanine, 3-fluoro-tyrosine,3-iodo-D-phenylalanine, 3-iodo-L-phenylalanine, 3-iodo-L-tyrosine,3-methoxy-L-tyrosine, 3-methyl-D-phenylalanine,3-methyl-L-phenylalanine, 3-nitro-D-phenylalanine,3-nitro-L-phenylalanine, 3-nitro-L-tyrosine,4-(trifluoromethyl)-D-phenylalanine,4-(trifluoromethyl)-L-phenylalanine, 4-amino-D-phenylalanine,4-amino-L-phenylalanine, 4-benzoyl-D-phenylalanine,4-benzoyl-L-phenylalanine, 4-bis(2-chloroethyl)amino-L-phenylalanine,4-bromo-D-phenylalanine, 4-bromo-L-phenylalanine,4-chloro-D-phenylalanine, 4-chloro-L-phenylalanine,4-cyano-D-phenylalanine, 4-cyano-L-phenylalanine,4-fluoro-D-phenylalanine, 4-fluoro-L-phenylalanine,4-iodo-D-phenylalanine, 4-iodo-L-phenylalanine, homophenylalanine,thyroxine, 3,3-diphenylalanine, thyronine, ethyl-tyrosine, andmethyl-tyrosine.

Amino acid analogs include analogs of proline. Examples of amino acidanalogs of proline include, but are not limited to, 3,4-dehydro-proline,4-fluoro-proline, cis-4-hydroxy-proline, thiazolidine-2-carboxylic acid,and trans-4-fluoro-proline.

Amino acid analogs include analogs of serine and threonine. Examples ofamino acid analogs of serine and threonine include, but are not limitedto, 3-amino-2-hydroxy-5-methylhexanoic acid,2-amino-3-hydroxy-4-methylpentanoic acid, 2-amino-3-ethoxybutanoic acid,2-amino-3-methoxybutanoic acid, 4-amino-3-hydroxy-6-methylheptanoicacid, 2-amino-3-benzyloxypropionic acid, 2-amino-3-benzyloxypropionicacid, 2-amino-3-ethoxypropionic acid, 4-amino-3-hydroxybutanoic acid,and α-methylserine.

Amino acid analogs include analogs of tryptophan. Examples of amino acidanalogs of tryptophan include, but are not limited to, the following:α-methyl-tryptophan; β-(3-benzothienyl)-D-alanine;β-(3-benzothienyl)-L-alanine; 1-methyl-tryptophan; 4-methyl-tryptophan;δ-benzyloxy-tryptophan; 5-bromo-tryptophan; 5-chloro-tryptophan;5-fluoro-tryptophan; 5-hydroxy-tryptophan; 5-hydroxy-L-tryptophan;5-methoxy-tryptophan; 5-methoxy-L-tryptophan; 5-methyl-tryptophan;6-bromo-tryptophan; 6-chloro-D-tryptophan; 6-chloro-tryptophan;6-fluoro-tryptophan; 6-methyl-tryptophan; 7-benzyloxy-tryptophan;7-bromo-tryptophan; 7-methyl-tryptophan;D-1,2,3,4-tetrahydro-norharman-3-carboxylic acid;6-methoxy-1,2,3,4-tetrahydronorharman-1-carboxylic acid;7-azatryptophan; L-1,2,3,4-tetrahydro-norharman-3-carboxylic acid;5-methoxy-2-methyl-tryptophan; and 6-chloro-L-tryptophan.

In some embodiments, amino acid analogs are racemic. In someembodiments, the D isomer of the amino acid analog is used. In someembodiments, the L isomer of the amino acid analog is used. In otherembodiments, the amino acid analog comprises chiral centers that are inthe R or S configuration. In still other embodiments, the amino group(s)of a β-amino acid analog is substituted with a protecting group, e.g.,tert-butyloxycarbonyl (BOC group), 9-fluorenylmethyloxycarbonyl (FMOC),tosyl, and the like. In yet other embodiments, the carboxylic acidfunctional group of a β-amino acid analog is protected, e.g., as itsester derivative. In some embodiments the salt of the amino acid analogis used.

A “non-essential” amino acid residue is a residue that can be alteredfrom the wild-type sequence of a polypeptide without abolishing orsubstantially altering its essential biological or biochemical activity(e.g., receptor binding or activation). An “essential” amino acidresidue is a residue that, when altered from the wild-type sequence ofthe polypeptide, results in abolishing or substantially abolishing thepolypeptide's essential biological or biochemical activity.

A “conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., K, R, H), acidic side chains (e.g., D, E), unchargedpolar side chains (e.g., G, N, Q, S, T, Y, C), nonpolar side chains(e.g., A, V, L, I, P, F, M, W), beta-branched side chains (e.g., T, V,I) and aromatic side chains (e.g., Y, F, W, H). Thus, a predictednonessential amino acid residue in a polypeptide, for example, isreplaced with another amino acid residue from the same side chainfamily. Other examples of acceptable substitutions are substitutionsbased on isosteric considerations (e.g. norleucine for methionine) orother properties (e.g. 2-thienylalanine for phenylalanine, or6-CI-tryptophan for tryptophan).

B. Detailed Description

The molecular cloning and characterization of Wnt3a was described bySaitoh et al., Biochem Biophys Res Commun, 2001, 29; 284(5):1168-75.Native human Wnt3a is a lipid modified growth factor, composed of 352amino acids (SEQ ID NO:1), that is effective in activating stem cells orstimulating their self-renewal. In some cases, due to its hydrophobicnature the active Wnt3a polypeptide or other Wnt polypeptides aredifficult to purify to homogeneity at scaleable levels. In one aspect,described herein is a method for purification of Wnt polypeptides. Insome instances, the purification method improves the purity and yield ofhuman Wnt polypeptides. In some embodiments, the purified Wntpolypeptide is used as part of a therapeutic regimen. In someembodiments, the purified Wnt polypeptide is used as a therapeuticpolypeptide.

As described elsewhere herein, Wnt polypeptides form a family of highlyconserved secreted signaling molecules. Wnt polypeptides can bemammalian, such as a human Wnt polypeptide. In some embodiments, Wntpolypeptides include Wnt1, Wnt2, Wnt2b (or Wnt13), Wnt3, Wnt3a, Wnt4,Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a (Wnt14, orWnt14b), Wnt9b (Wnt14b, or Wnt15), Wnt10a, Wnt10b (or Wnt12), Wnt11,Wnt-16a, or a Wnt-16b polypeptide sequences or a functionally activevariants therof. In some embodiments, a Wnt polypeptide is selected fromthe group consisting of a Wnt3a polypeptide, Wnt5a polypeptide, Wnt10bpolypeptide and a functionally active Wnt variants therof In someembodiments, the Wnt polypeptide is a Wnt3a polypeptide or afunctionally active variant therof. In some embodiments, the Wntpolypeptide is a Wnt5a polypeptide or a functionally active varianttherof. In some embodiments, the Wnt polypeptide is a Wnt10b polypeptideor a functionally active variant therof. In some embodiments, the Wnt3apolypeptide is a human Wnt3a polypeptide or a functionally activevariant therof. In some instances, the sequence identity of afunctionally active variant Wnt3a polypeptide is at least about 30%,about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about95%, or about 99% identical to a native Wnt3a polypeptide sequence, suchas a human Wnt3a polypeptide sequence (e.g., SEQ ID NO:1). In someinstances, the sequence identity of a functionally active variant Wnt3apolypeptide is at most about 30%, about 40%, about 50%, about 60%, about70%, about 80%, about 90%, about 95%, or about 100% identical to anative Wnt3a polypeptide sequence, such as a human Wnt3a polypeptidesequence (e.g., SEQ ID NO:1). In some instances, the amino acid sequenceof the Wnt3a polypeptide is as set forth in SEQ ID NO:1, or the Wnt3apolypeptide is encoded by the nucleic acid sequence of SEQ ID NO:2.

In some instances, the purification scheme described herein is simple,and inexpensive. In some cases, the purification scheme described hereinenables purification of an functionally active form of Wnt polypeptidewith higher purity. In some embodiments, the purification methodsdescribed herein enable purification of Wnt polypeptides from culturemedia of recombinant host cells. In some instances, the recombinant hostcell or the expression cell line is a prokaryotic host cell orexpression cell line, a yeast host cell or expression cell line, aninsect host cell or expression cell line, or a mammalian host cell orexpression cell line.

In some embodiments, the recombinant host cell or expression cell lineis a yeast host cell or expression cell line. Exemplary yeast host cellsinclude, but are not limited to, Pichia pastoris yeast strains such asGS115, KM71H, SMD1168, SMD1168H, and X-33; and Saccharomyces cerevisiaeyeast strain such as INVSc1.

In some embodiments, the recombinant host cell or expression cell lineis an insect host cell or expression cell line. Exemplary insect celllines include, but are not limited to, Drosophila S2 cells, Sf9 cells,Sf21 cells, High Five™ cells, and expresSF+® cells.

In some embodiments, the recombinant host cell or expression cell lineis a mammalian host cell or expression cell line. In some cases,mammalian cell lines is a stable cell lines, or a cell line that hasincorporated a genetic material of interest into its own genome and hasthe capability to express the products of the genetic material aftermany generations of cell division. In some embodiments, the mammaliancell line is a Current Good Manufacturing Practices (cGMP) compliantcell line. Exemplary mammalian cell lines include, but are not limitedto, 293A cell line, 293FT cell line, 293F cells, 293 H cells, CHO DG44cells, CHO-S cells, CHO-K1 cells, Expi293F™ cells, Flp-In™ T-REx™ 293cell line, Flp-In™-293 cell line, Flp-In™-3T3 cell line, Flp-In™-BHKcell line, Flp-In™-CHO cell line, Flp-In™-CV-1 cell line, Flp-In™-Jurkatcell line, FreeStyle™ 293-F cells, FreeStyle™ CHO-S cells, GripTite™ 293MSR cell line, GS-CHO cell line, HepaRG™ cells, T-REx™ Jurkat cell line,Per.C6 cells, T-REx™-293 cell line, T-REx™-CHO cell line, andT-REx™-HeLa cell line. In some embodiments, the mammalian cell line is aCHO cell line. In some embodiments, the mammalian cell line is a CHO-Scell line. As used herein, “CHO cells” or “CHO cell line” is a genericterm that encompasses the different types of CHO cells and CHO celllines.

In some embodiments, the mammalian host cells are grown as a liquidsuspension culture, in soft agar, on top of soft agar, or as monolayer.In some embodiments, the CHO cells are grown as a liquid suspensionculture, in soft agar, on top of soft agar, or as monolayer. In someembodiments, the CHO cells are cultured in liquid suspension culture. Insome cases, the culture medium contains a serum. Non-limiting examplesof serum include fetal bovine serum (FBS), HyClone FetalClone II andIII, iron-supplemented bovine calf serum (ICS), and human plateletlysates. In some embodiments, the serum is FBS. In some embodiments, theFBS presents in the medium is at most about 0.1%, 0.5%, 1%, 2%, 3%, 4%,5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, or less. In someembodiments, the FBS presents in the medium is at least about 0.1%,0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%,or more.

In some embodiments, the serum (e.g. FBS) is needed for the induction ofexpression and for secretion of Wnt polypeptides (e.g. Wnt3apolypeptide) into the culture medium. In some embodiments, the serumprovides one or more essential factors that are used for lipidmodification on a Wnt polypeptide (e.g. residues such as Ser 209 inWnt3a). In some embodiments, the serum provides one or more chaperoneproteins and lipophilic molecules that are used for the transportationof Wnt polypeptide (e.g. Wnt3a) from the endoplasmic reticulum (ER) tothe Golgi and then to the cell membrane.

In some embodiments, the serum is modified to remove the lipidcomponents. In some instances, a defined set of lipids is thenintroduced into the modified serum. In some cases, a stripping agent,such as charcoal, is used to remove non-polar lipophilic materials (e.g.viruses, growth factors, and hormones) from the serum. In someinstances, a lipid supplement is introduced into the modified serum.Non-limiting examples of lipid supplement include Lipid Mixture 1(Sigma-Aldrich), Lipid Mixture 2 (Sigma-Aldrich), Lipogro® (RockyMountain Biologicals), and Chemically Defined Lipid Concentration (LifeTechnologies).

In some embodiments, the mammalian host cells (e.g. CHO or CHO-S cells)are cultured in a serum-free medium. Non-limiting examples of serum-freemedia include CD CHO medium, CD CHO AGT™ medium, CD OptiCHO™ medium,CHO-S—SFM II (optionally including hypoxanthine and thymidine), CD 293AGT™ medium, Adenovirus Expression Medium (AEM), FreeStyle™ 293Expression medium, EX-CELL® 302 Serum-Free medium, EX-CELL® 325 PF CHOSerum-Free medium, EX-CELL® CD CHO-2 medium animal-component free,EX-CELL® CD CHO-3 medium, and EX-CELL® CDHO DHFR⁻ mediumanimal-component free.

In some embodiments, the serum-free medium contains one or moreadditional supplement. In some embodiments, the additional supplement isa serum. In some cases, the serum is FBS. In some cases, the FBSpresents in the serum-free medium is at most about 0.1%, 0.5%, 1%, 2%,3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, or less. Insome cases, the FBS presents in the serum-free medium is at least about0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%,15%, or more.

In some embodiments, the additional supplement is a lipid supplement.Non-limiting examples of lipid supplement include Lipid Mixture 1(Sigma-Aldrich), Lipid Mixture 2 (Sigma-Aldrich), Lipogro® (RockyMountain Biologicals), and Chemically Defined Lipid Concentration (LifeTechnologies). In some embodiments, the serum-free medium contains alipid supplement.

In some embodiments, the additional supplement is a serum substitute. Insome embodiments, the serum substitute is selected from EXCYTE(Millipore), EX-CYTE+human serum albumin+AOF ITS (Insulin, transferrin,and selenium, Millipore), Human serum albumin (Millipore), Heparinsulfate (Sigma), lipids such as described elsewhere herein and notlimiting to DMPC and cholesterol, Cell-ess (Essential Pharmaceuticals),and the like. In some instances, the Wnt polypeptide in the presence ofa serum substitute is secreted into the conditioned medium. In someinstances, the Wnt polypeptide in the presence of a serum substitute isnot secreted into the conditioned medium. In some instances, the Wnt3apolypeptide in the presence of a serum substitute is secreted into theconditioned medium. In some instances, the Wnt3a polypeptide in thepresence of a serum substitute is not secreted into the conditionedmedium.

In some embodiments, an expression vector that is tolerant of aserum-free medium condition is used. In some cases, the expressionvector leads to a high copy number of the desired transcript andsecretion of the polypeptide of interest. In some instances, theexpression vector is compatible with cGMP compatible mammalian celllines. Non-limiting examples of mammalian expression vectors includepOptivec vector, pTargeT™ vector, BacMam pCMV-Dest vector, Flp-In™ coresystem, Gateway® suite of vectors, HaloTag® vector, Flexi® vector,pCMVTNT™ vector, and pcDNA™4/TO vector. In some embodiments, theexpression vector is selected from pOptivec and pTargeT™ vectors. ThepOptivec vector is a TOPO® adapted bicistronic plasmid which allowsrapid cloning of a gene containing a mammalian secretion signal and thegene of interest downstream of the CMV promoter. The dihydrofolatereductase selection markers allows for rapid selection. In some cases,this vector is used for transient transfection of CHO-S cells. In someinstances, the pTargeT™ vector is used for transient transfection ofCHO-S cells and for creating a stable cell line expressing a Wntpolypeptide (e.g. Wnt3a).

In some embodiments, acidification of the culture condition is used toaid in Wnt secretion. In some instances, acidification of the culturecondition is used to aid in Wnt3a secretion. In some instances,acidification mimics vesicular acidification. In some instances,acidification is an acidification of the cytoplasm. In some instances,acidification occurs naturally such as acids secreted by the cellsgrowning in the conditioned media, or artifically such as the additionof acids such as for example acetic acid. In some instances,acidification aids in secretion of Wnt polypeptide. In some instances,acidification aids in secretion of Wnt3a polypeptide.

In some embodiments, the expression method of a Wnt polypeptide in yeastcells, insect cells, or mammalian cells follows protocols well known inthe art. In some instances, the Wnt polypeptide is expressed inmammalian cells. In some embodiments, a transcriptional induction agentis used to induce expression of the Wnt polypeptide. In some cases, thetranscriptional induction agent includes doxycycline, tetracycline, andcoumermycin. In some cases, the expression is constitutive, or that thepolypeptide is expressed without an induction agent. In someembodiments, the Wnt polypeptide is secreted into the medium. In someinstances, the conditioned medium is harvested and an additional agentis added to solubilize the Wnt polypeptide. In some instances, theadditional agent is a detergent. In some instances, the detergent is anonionic detergent, an anionic detergent, or a zwitterionic detergent.Exemplary detergents include, but are not limited to, Brij-35, Brij-58,3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS),3-([3-Cholamidopropyl]dimethylammonio)-2-hydroxy-1-propanesulfonate(CHAPSO), nonyl phenoxypolethoxylethanol (NP-40), Octyl glucoside, Octylthioglucoside, sodium dodecyl sulfate (SDS), Triton X-100, Triton X-114,Tween 20, and Tween 80. In some embodiments, the detergent is CHAPS orTriton X-100. In some embodiments, the detergent is CHAPS. CHAPS is azwitterionic detergent useful for membrane protein solubilization. Insome embodiments, the Wnt polypeptide is also post-translationallymodified by glycosylation and palmitoylation.

Palmitoylation is covalent attachment of fatty acids, such as palmiticacid, to cysteine, serin, and threonine residues. In some instances,palmitoylation enhances the hydrophobicity of proteins and contribute totheir membrane association. For example, Nusse et al. reported that themurine Wnt3a protein was S-palmitoylated at a conserved cysteine residue(C77) (Willert et al., Nature, 2003, 423:448-452) and that a mutant formof mouse Wnt3a protein, wherein the palmitoylated Cys77 was substitutedwith alanine (C77A), showed a diminished ability to activate Wntsignaling, but was secreted normally into the culture medium. Further,the study showed that palmitoylation of the Cys77 residue was importantfor biological activity.

In some embodiments, the Wnt polypeptide is palmitoylated at a cysteine,serine, and/or threonine residue. In some embodiments, the Wnt3apolypeptide is palmitoylated at a cysteine, serine, and/or threonineresidue. In some instances, the Wnt3a polypeptide is palmitoylated atC77, S139, S181, S209, or S211, as set forth in SEQ ID NO:1. In someinstances, the Wnt3a polypeptide is palmitoylated at one or more of theamino acid positions selected from C77, S139, S181, S209, or S211, asset forth in SEQ ID NO:1. In some cases, the Wnt3a polypeptide ispalmitoylated at C77, as set forth in SEQ ID NO:1. In some cases, theWnt3a polypeptide is palmitoylated at S209, as set forth in SEQ ID NO:1.In some cases, the Wnt3a polypeptide is palmitoylated at C77 and atS209, as set forth in SEQ ID NO:1. In some cases, the Wnt3a protein isnot palmitoylated at C77, as set forth in SEQ ID NO:1. In some cases,the Wnt3a protein is palmitoylated at S209 and is not palmitoylated atC77, as set forth in SEQ ID NO:1.

In some embodiments, the Wnt polypeptide comprising palmitoylatedresidues are functionally active variant polypeptides, such as afunctionally active variant of a Wnt3a native polypeptide (e.g., humanWnt3a). In some embodiments, the Wnt3a functionally active variantpolypeptide comprises palmitoylated residues selected from C77, S139,S181, S209, and S211, as set forth in SEQ ID NO:1. In some embodiments,the Wnt3a functionally active variant polypeptide has a palmitoylatedresidue at S139 as set forth in SEQ ID NO:1. In some embodiments, thefunctionally active variant Wnt3a polypeptide has a palmitoylatedresidue at S181 as set forth in SEQ ID NO:1. In some embodiments, thefunctionally active variant Wnt3a polypeptide has a palmitoylatedresidue at S209 as set forth in SEQ ID NO:1 In some embodiments, thefunctionally active variant Wnt3a polypeptide has a palmitoylatedresidue at S211 as set forth in SEQ ID NO:1. In some embodiments, thefunctionally active variant Wnt3a polypeptide has a palmitoylatedresidue at S209 and one other residue as set forth in SEQ ID NO:1. Insome embodiments, the one other residue does not include C77. In someembodiments, the functionally active variant Wnt3a polypeptide ispalmitoylated at S209 and is not palmitoylated at C77, as set forth inSEQ ID NO:1.

In some embodiments, the purification scheme described herein pertainsto a Wnt polypeptide selected from Wnt1, Wnt2, Wnt2b (or Wnt13), Wnt3,Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a(Wnt14, or Wnt14b), Wnt9b (Wnt14b, or Wnt15), Wnt10a, Wnt10b (or Wnt12),Wnt11, Wnt-16a, and Wnt-16b polypeptide. In some instances, thepurification scheme described herein pertains to Wnt3a polypeptide, aWnt5a polypeptide, or a Wnt10b polypeptide. In some cases, thepurification scheme described herein pertains to a Wnt3a polypeptide. Insome cases, the purification scheme described herein pertains to a Wnt5apolypeptide. In some cases, the purification scheme described hereinpertains to a Wnt10b polypeptide.

In some embodiments, the purification scheme described herein pertainsto a Wnt3a polypeptide that has palmitoylation at a cysteine residuethat correspond to the cysteine at amino acid position 77 as set forthin SEQ ID NO:1 or a serine residue that correspond to the serine atamino acid position 209 as set forth in SEQ ID NO:1. In someembodiments, the purification scheme described herein selects a Wnt3apolypeptide that has palmitoylation at the cysteine residue thatcorrespond to the cysteine at amino acid position 77 as set forth in SEQID NO:1. In some embodiments, the purification scheme described hereinselects a Wnt3a polypeptide that has palmitoylation at the serineresidue that correspond to the serine at amino acid position 209 as setforth in SEQ ID NO:1. In some embodiments, the purification schemedescribed herein selects a Wnt3a polypeptide that has palmitoylation atthe serine residue that correspond to the serine at amino acid position209 as set forth in SEQ ID NO:1 and at least one other palmitoylatedresidue. In some embodiments, the at least one other palmitoylatedresidue is not C77 as set forth in SEQ ID NO:1. In some embodiments, thepurification scheme described herein does not select a Wnt3a polypeptidethat has palmitoylation at the cysteine residue that correspond to thecysteine at amino acid position 77 as set forth in SEQ ID NO:1.

In some embodiments, the purification scheme described herein utilizes aseparation step. In some embodiments, the separation step is anion-exchange purification step, a hydrophobic purification step, or anaffinity purification step. In some cases, the separation step is anion-exchange purification step. In some instances, the ion-exchangepurification step utilizes beads immobilized with one or more sulfonatedpolyaromatic compounds. In some instances, the ion-exchange purificationstep utilizes a column immobilized with one or more sulfonatedpolyaromatic compounds. A non-limiting example of a sulfonatedpolyaromatic compound is Cibacron blue F3GA. In some instances, Cibacronblue F3GA is a triazinyl dye. In some instances, beads and/or columnsimmobilized with a triazinyl dye is used during the ion-exchangepurification step. A non-limiting example of a chromatographic columnimmobilized with Cibacron blue F3GA is a Blue Sepharose column.

In some embodiments, purification is carried out in batch mode with theuse of beads immobilized with a sulfonated polyaromatic compound. Ingeneral, the Wnt polypeptide is bound to the sulfonated polyaromaticcompound immobilized beads in a binding buffer containing a lowconcentration of salt. High salt destabilizes the non-covalent ionicinteractions between protein and the beads, thereby allow elution of theWnt polypeptide. In some embodiments, the concentration of the salt usedin the binding buffer is at most 0, 0.01, 5, 10, 15, 20, 25, 30, 40, 50mM, or less. In some embodiments, the concentration of the salt used inthe binding buffer is at least 0, 0.01, 5, 10, 15, 20, 25, 30, 40, 50mM, or more. In some embodiments, one or more wash buffers are used toremove unbound impurities. In some embodiments, at most 1, 2, 3, 4, 5,or more wash steps are used. In some embodiments, at least 1, 2, 3, 4, 5or less wash steps are used. In some embodiments, the concentration ofthe salt used in the wash buffer is at least 30, 40, 50, 60, 70, 80, 90,100 mM, more. In some embodiments, the concentration of the salt used inthe wash buffer is at most 30, 40, 50, 60, 70, 80, 90, 100 mM, less. Insome embodiments, one or more elution steps follow. In some embodiments,the concentration of the salt in the elution buffer is at least 80, 90,100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350,400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500,2000 mM, or more. In some embodiments, the concentration of the salt inthe elution buffer is at most 80, 90, 100, 110, 120, 130, 140, 150, 160,170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700,750, 800, 850, 900, 950, 1000, 1500, 2000 mM, or less. Exemplary saltinclude sodium chloride, potassium chloride, magnesium chloride, calciumchloride, calcium phosphate, potassium phosphate, magnesium phosphate,sodium phosphate, ammonium sulfate, ammonium chloride, ammoniumphosphate, and the like. In some embodiments, a detergent is alsoformulated into the binding buffer, wash buffer, and/or elution buffer.In some embodiments, the detergent is CHAPS or Triton X-100. In someembodiments, the percentage of CHAPS or Triton X-100 is at least 0.01%,0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, or more. In someembodiments, the percentage of CHAPS or Triton X-100 is at most 0.01%,0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, or less. In someinstances, buffer components such as tris(hydroxymethyl)methylamine HCl(Tris-HCl), 3-{[tris(hydroxymethyl)methyl]amino}propanesulfonic acid(TAPS), N,N-bis(2-hydroxyethyl)glycine (Bicine),N-tris(hydroxymethyl)methylglycine (Tricine),3-[N-Tris(hydroxmethyl)methylamino]-2-hydroxypropanesulfonic acid(TAPSO), 4-2-hydroxyethyl-1-piperazineethanesulfonic acid (HEPES),3-(N-morpholino)propanesulfonic acid (MOPS),piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES),2-(N-morpholino)ethanesulfonic acid (MES), and the like, are used. Insome instances, the pH of the buffer is at least 4, 4.5, 5, 5.5, 6, 6.5,7, 7.5, 8, 8.5, 9, or more. In some instances, the pH of the buffer isat most 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, or less. In someembodiments, the Wnt polypeptide is Wnt3a polypeptide, Wnt5apolypeptide, or Wnt10b polypeptide. In some embodiments, the Wntpolypeptide is Wnt3a polypeptide.

In some embodiments, purification is carried out using a columnimmobilized with a sulfonated polyaromatic compound. In general, the Wntpolypeptide is bound to the column immobilized with the sulfonatedpolyaromatic compound in a binding buffer containing a low concentrationof salt. High salt destabilizes the non-covalent ionic interactionsbetween the polypeptide and the column beads, thereby allow elution ofthe Wnt polypeptide. In some embodiments, the concentration of the saltused in the binding buffer is at most 0, 0.01, 5, 10, 15, 20, 25, 30,40, 50 mM, or less. In some embodiments, the concentration of the saltused in the binding buffer is at least 0, 0.01, 5, 10, 15, 20, 25, 30,40, 50 mM, or more. In some embodiments, one or more wash buffers areused to remove unbound impurities. In some embodiments, at most 1, 2, 3,4, 5, or more wash steps are used. In some embodiments, at least 1, 2,3, 4, 5 or less wash steps are used. In some embodiments, theconcentration of the salt used in the wash buffer is at least 30, 40,50, 60, 70, 80, 90, 100 mM, more. In some embodiments, the concentrationof the salt used in the wash buffer is at most 30, 40, 50, 60, 70, 80,90, 100 mM, less. In some embodiments, one or more elution steps follow.In some embodiments, the concentration of the salt in the elution bufferis at least 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190,200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850,900, 950, 1000, 1500, 2000 mM, or more. In some embodiments, theconcentration of the salt in the elution buffer is at most 80, 90, 100,110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400,450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000mM, or less. Exemplary salt include sodium chloride, potassium chloride,magnesium chloride, calcium chloride, calcium phosphate, potassiumphosphate, magnesium phosphate, sodium phosphate, ammonium sulfate,ammonium chloride, ammonium phosphate, and the like. In someembodiments, a detergent is also formulated into the binding buffer,wash buffer, and/or elution buffer. In some embodiments, the detergentis CHAPS or Triton X-100. In some embodiments, the percentage of CHAPSor Triton X-100 is at least 0.01%, 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%,3.5%, 4%, 4.5%, 5%, or more. In some embodiments, the percentage ofCHAPS or Triton X-100 is at most 0.01%, 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%,3%, 3.5%, 4%, 4.5%, 5%, or less. In some instances, buffer componentssuch as tris(hydroxymethyl)methylamine HCl (Tris-HCl),3-{[tris(hydroxymethyl)methyl]amino}propanesulfonic acid (TAPS),N,N-bis(2-hydroxyethyl)glycine (Bicine),N-tris(hydroxymethyl)methylglycine (Tricine),3-[N-Tris(hydroxymethyl)methylamino]-2-hydroxypropanesulfonic acid(TAPSO), 4-2-hydroxyethyl-1-piperazineethanesulfonic acid (HEPES),3-(N-morpholino)propanesulfonic acid (MOPS),piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES),2-(N-morpholino)ethanesulfonic acid (MES), and the like, are used. Insome instances, the pH of the buffer is at least 4, 4.5, 5, 5.5, 6, 6.5,7, 7.5, 8, 8.5, 9, or more. In some instances, the pH of the buffer isat most 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, or less. In someembodiments, the Wnt polypeptide is Wnt3a polypeptide, Wnt5apolypeptide, or Wnt10b polypeptide. In some embodiments, the Wntpolypeptide is Wnt3a polypeptide.

In some embodiments, the separation step is a hydrophobic purificationstep. In some embodiments, the hydrophobic purification step utilizeseither beads immobilized with a hydrophobic ligand or a columnimmobilized with a hydrophobic ligand. Non-limiting examples of theligands include butyl, octyl, phenyl, Protein A, and the like.

In some embodiments, the separation step is an affinity purificationstep. In some embodiments, the affinity purification step utilizeseither beads immobilized with an affinity ligand or a column immobilizedwith an affinity ligand. Exemplary ligands include Frizzled receptor(Fzd) or its fragments thereof, and low-density lipoproteinreceptor-related protein 6 (LRP6), or its fragments thereof.

In some embodiments, the concentration and yield of the eluted Wntpolypeptide is measured prior to subjecting to a further purificationstep. In some embodiments, the yield is at least about 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 95%, or more. In some embodiments, the yield isat most about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or less. Insome embodiments, the purity is at least about 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 95%, or more. In some embodiments, the purity is at mostabout 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or less.

In some embodiments, the purified Wnt polypeptide is Wnt3a polypeptide.In some embodiments, the Wnt3a polypeptide containing a palmitoylationat an amino acid residue corresponding to Serine 209 as set forth in SEQID NO:1 is purified from the separation step. In some embodiments, theWnt3a polypeptide containing a palmitoylation at an amino acid residuecorresponding to Cysteine 77 as set forth in SEQ ID NO:1 is purifiedfrom the separation step. In some embodiments, the Wnt3a polypeptidecontaining a palmitoylation at an amino acid residue corresponding toCysteine 77 and a palmitoylation at an amino acid residue correspondingto Serine 209 as set forth in SEQ ID NO:1 is purified from theseparation step. In some embodiments, the Wnt3a polypeptide containing apalmitoylation at an amino acid residue corresponding to Serine 209 asset forth in SEQ ID NO:1 and a palmitoylation at at least one otheramino acid residue is purified from the separation step. In someembodiments, the at least one other amino acid residue is not C77 as setforth in SEQ ID NO:1.

In some embodiments, the concentration of the Wnt3a specie containingthe Serine 209 palmitoylation is high in the eluted product than theWnt3a specie containing the Cysteine 77 palmitoylation. In someembodiments, the concentration of the two Wnt3a species is defined by avalue, such as a ratio. In some embodiments, the ratio of the Wnt3aSerine 209 specie to the Wnt3a Cysteine 77 specie is between about50:50, about 55:45, about 60:40, about 65:35, about 70:30, about 75:25,about 80:20, about 85:15, about 90:10, about 95:5, about 99:1, or about100:0. In some embodiments, the concentration of the Wnt3a speciecontaining the Serine 209 palmitoylation is high in the eluted productthan the Wnt3a specie containing the Cysteine 77 and the Serine 209palmitoylations. In some embodiments, the concentration of the two Wnt3aspecies is defined by a value, such as a ratio. In some embodiments, theratio of the Wnt3a Serine 209 specie to the Wnt3a Cysteine 77/Serine 209specie is between about 50:50, about 55:45, about 60:40, about 65:35,about 70:30, about 75:25, about 80:20, about 85:15, about 90:10, about95:5, about 99:1, or about 100:0.

In some embodiments, the further purification step is a step thatutilizes pre-fabricated liposomes. In some embodiments, the liposomeeliminates the serum (FBS) from the polypeptide preparation. In someembodiments, this latter step takes advantage of the hydrophobicity ofthe Wnt polypeptide, and eliminates the need for additional orsubsequent purification steps, such as for example, subsequent gelfiltration and/or chromatographic purification steps (e.g. heparinsulfate immobilized columns).

In some embodiments, the liposome is fabricated using methods well knownin the art. Liposomes are artificially-prepared spherical vesicles thatcompose a lamellar phase lipid bilayer and an aqueous core. There areseveral types of liposomes, such as the multilamellar vesicle (MLV),small unilamellar liposome vesicle (SUV), the large unilamellar vesicle(LUV), and the cochleate vesicle. In some instances, liposomes areformed by phospholipids. In some embodiments, phospholipids areseparated into those with diacylglyceride structures or those derivedfrom phosphosphingolipids. In some embodiments, the diacylglyceridestructures include phosphatidic acid (phosphatidate) (PA),phosphatidylethanolamine (cephalin) (PE), phosphatidylcholine (lecithin)(PC), phosphatidylserine (PS), and phosphoinositides such asphosphatidylinositol (PI), phosphatidylinositol phosphate (PIP),phosphatidylinositol bisphosphate (PIP2), and phosphatidylinositoltriphosphate (PIP3). In some embodiments, phosphosphingolipids includeceramide phosphorylcholine, ceramide phosphorylethanolamine, andceramide phosphoryllipid. In some embodiments, the liposomes are formedfrom phosphatidylcholines.

In some embodiments, the lipids are also selected based on itstransition phase temperature (T_(m)), or the temperature interfacebetween the liquid crystalline phase and the gel phase. In someembodiments, the T_(m) is governed by the head group species,hydrocarbone length, unsaturation, and the charge. For example, shortlipids (lipids containing 8, 10, or 12 tail carbon chain length) haveliquid crystalline phase at temperatures below 4° C. However, liposomesmanufactured from these short chain carbon lipids are toxic to cellsbecause they dissolve cell membranes. Liposomes manufactured from longercarbon-chain lipids are not toxic to cells, but their transitiontemperatures are higher. For example,1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) which has a 16 tailcarbon length, has a T_(m) of about 41° C. In some embodiments, thelipids used herein have a T_(m) of between about 10° C. and about 37°C., 15° C. and about 30° C., 18° C. and about 27° C., or 21° C. andabout 25° C. In some embodiments, the lipids used herein have a T_(m) ofat least 22° C., 23° C., 24° C., or more. In some embodiments, thelipids used herein have a T_(m) of at most 22° C., 23° C., 24° C., orless. In some embodiments, the lipids used herein have a tail carbonlength of at least about 12, 13, 14, or more. In some embodiments, thelipids used herein have a tail carbon length of at most about 12, 13,14, or less.

In some embodiments, the lipids are further selected based on the netcharge of the liposome. In some embodiments, the liposome has a netcharge of 0 at a pH of between about 4.0 and about 10.0, about 5.0 andabout 9.0, about 6.5 and about 8.0, about 7.0 and about 7.8, or about7.2 and about 7.6. In some embodiments, the liposome has a net charge of0 at a pH of about 7.3, about 7.4, or about 7.5. In some embodiments,the liposome has a net positive charge at a pH of between about 4.0 andabout 10.0, about 5.0 and about 9.0, about 6.5 and about 8.0, about 7.0and about 7.8, or about 7.2 and about 7.6. In some embodiments, theliposome has a net positive charge at a pH of about 7.3, about 7.4, orabout 7.5. In some embodiments, the liposome has a net negative chargeat a pH of between about 4.0 and about 10.0, about 5.0 and about 9.0,about 6.5 and about 8.0, about 7.0 and about 7.8, or about 7.2 and about7.6. In some embodiments, the liposome has a net negative charge at a pHof about 7.3, about 7.4, or about 7.5.

In some embodiments, lipids are selected from1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC),1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),1-tetradecanoyl-2-hexadecanoyl-sn-glycero-3-phosphocholine (MPPC),1,2-dimyristoyl-sn-glycero-3-phospho-L-serine (DMPS), and1,2-dihexanoyl-sn-glycero-3-phosphocholine (DMPG). In some embodiments,the lipid is DMPC.

In some embodiments, an additional lipid is fabricated into theliposome. In some embodiments, the additional lipid is cholesterol. Insome instances, the concentration of a phosphatidylcholine such as DMPCand cholesterol is defined by a value such as a ratio. In someembodiments, the ratio of the concentrations of phosphatidylcholine suchas DMPC and cholesterol is between about 50:50, about 55:45, about60:40, about 65:35, about 70:30, about 75:25, about 80:20, about 85:15,about 90:10, about 95:5, about 99:1, or about 100:0. In someembodiments, the ratio of the concentrations of phosphatidylcholine suchas DMPC and cholesterol is about 90:10. In some embodiments, theconcentration unit is moles. In some embodiments, the ratio ismole:mole.

In some embodiments, the Wnt polypeptide is reconstituted with aliposome at a concentration of at least about 0.01, 0.015, 0.02, 0.025,0.03, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08,0.085, 0.09, 0.095, 0.1, 0.15, 0.2, 0.25, 0.3, 0.4, 0.5 ng/μL or more.In some embodiments, the Wnt polypeptide is reconstituted with aliposome at a concentration of at most about 0.01, 0.015, 0.02, 0.025,0.03, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08,0.085, 0.09, 0.095, 0.1, 0.15, 0.2, 0.25, 0.3, 0.4, 0.5 ng/μL or less.In some embodiments, the Wnt polypeptide is Wnt3a polypeptide, Wnt5apolypeptide, or Wnt10b polypeptide. In some embodiments, the Wntpolypeptide is Wnt3a polypeptide.

In some embodiments, the Wnt polypeptide is reconstituted with aliposome at a ratio of at least about 0.1:50, 0.5:30, 1:20, or 1:14 Wntpolypeptide to liposome, or more. In some embodiments, the Wntpolypeptide is reconstituted with a liposome at a ratio of at most about0.1:50, 0.5:30, 1:20, or 1:14 Wnt polypeptide to liposome, or less. Insome instances, the ratio is a weight to weight ratio. In someinstances, the unit of Wnt polypeptide is nanogram unit.

In some embodiments, the temperature at which the Wnt polypeptide isreconstituted with a liposome is at least between about 15° C. and about37° C., about 18° C. and about 33° C., or about 20° C. and about 28° C.In some embodiments, the temperature is at least about 21° C., 22° C.,23° C., 24° C., 25° C., 26° C., 27° C., or more. In some embodiments,the temperature is at most about 21° C., 22° C., 23° C., 24° C., 25° C.,26° C., 27° C., or less. In some embodiments, the Wnt polypeptide isWnt3a polypeptide, Wnt5a polypeptide, or Wnt10b polypeptide. In someembodiments, the Wnt polypeptide is Wnt3a polypeptide.

In some embodiments, the Wnt polypeptide is integrated into theliposomal membrane. In some cases, the Wnt polypeptide protrudes fromthe liposomal membrane onto the surface of the lipid membrane. In someinstances, the Wnt polypeptide is not incorporated into the aqueous coreof the liposome. In some embodiments, the Wnt polypeptide is Wnt3apolypeptide, Wnt5a polypeptide, or Wnt10b polypeptide. In someembodiments, the Wnt polypeptide is Wnt3a polypeptide. In someembodiments, the Wnt3a polypeptide is integrated into the liposomalmembrane. In some cases, the Wnt3a polypeptide protrudes from theliposomal membrane onto the surface of the lipid membrane. In someinstances, the Wnt3a polypeptide is not incorporated into the aqueouscore of the liposome.

In some embodiments, the Wnt polypeptide reconstituted with a liposomeis referred to as liposomal Wnt polypeptide or L-Wnt. In someembodiments, the Wnt polypeptide is Wnt3a polypeptide, Wnt5apolypeptide, or Wnt10b polypeptide. In some embodiments, the Wntpolypeptide is Wnt3a polypeptide. In some embodiments, the Wnt3apolypeptide reconstituted with a liposome is referred to as liposomalWnt3a polypeptide or L-Wnt3a. In some embodiments, the Wnt polypeptideis Wnt5a polypeptide. In some embodiments, the Wnt5a polypeptidereconstituted with a liposome is referred to as liposomal Wnt5apolypeptide or L-Wnt5a. In some embodiments, the Wnt polypeptide isWnt10b polypeptide. In some embodiments, the Wnt10b polypeptidereconstituted with a liposome is referred to as liposomal Wnt10bpolypeptide or L-Wnt10b.

In some embodiments, the L-Wnt undergoes a centrifugation step and isthen suspended in a buffer such as phosphate buffered saline (PBS). Insome instances, the L-Wnt is stored under nitrogen. In some instances,the L-Wnt is stable under nitrogen without substantial loss of activity.In some instances, the L-Wnt is stored at a temperature of between about1° C. and about 8° C. In some instances, the L-Wnt is stable at atemperature of at least about 1° C., 2° C., 3° C., 4° C., 5° C., 6° C.,7° C., 8° C., or more without substantial loss of activity. In someinstances, the L-Wnt is stable at a temperature of at most about 1° C.,2° C., 3° C., 4° C., 5° C., 6° C., 7° C., 8° C., or less withoutsubstantial loss of activity. In some embodiments, the L-Wnt is stablefor at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,75, 80, 85, 90, 95, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109,110, 115, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 356, 400,700, 1000 days, or more without substantial loss of activity. In someembodiments, the L-Wnt is stable for at most about 10, 15, 20, 25, 30,35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 101, 102, 103,104, 105, 106, 107, 108, 109, 110, 115, 120, 130, 140, 150, 160, 170,180, 190, 200, 300, 356, 400, 700, 1000 days, or less withoutsubstantial loss of activity.

In some embodiments, the L-Wnt3a undergoes a centrifugation step and isthen suspended in a buffer such as phosphate buffered saline (PBS). Insome instances, the L-Wnt3a is stored under nitrogen. In some instances,the L-Wnt3a is stable under nitrogen without substantial loss ofactivity. In some instances, the L-Wnt3a is stored at a temperature ofbetween about 1° C. and about 8° C. In some instances, the L-Wnt3a isstable at a temperature of at least about 1° C., 2° C., 3° C., 4° C., 5°C., 6° C., 7° C., 8° C., or more without substantial loss of activity.In some instances, the L-Wnt3a is stable at a temperature of at mostabout 1° C., 2° C., 3° C., 4° C., 5° C., 6° C., 7° C., 8° C., or lesswithout substantial loss of activity. In some embodiments, the L-Wnt3ais stable for at least about 10, 20, 30, 40, 50, 60, 70, 80 90, 100,110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 356, 400, 700,1000 days, or more without substantial loss of activity. In someembodiments, the L-Wnt3a is stable for at most about 10, 20, 30, 40, 50,60, 70, 80 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200,300, 356, 400, 700, 1000 days, or less without substantial loss ofactivity.

In some instances, the term “without substantial loss of activity”refers to the functional activity of a liposomal Wnt polypeptide is nearto that of the corresponding native Wnt polypeptide in the absence of aliposome. In some instances, the functional activity of the liposomalWnt polypeptide is at least about 100%, 99%, 95%, 90%, 85%, 80%, 75%,70%, 60%, 50%, 40%, or more compared to the functional activity of thenative Wnt polypeptide. In some instances, the functional activity ofthe liposomal Wnt polypeptide is at most about 100%, 99%, 95%, 90%, 85%,80%, 75%, 70%, 60%, 50%, 40%, or less compared to the functionalactivity of the native Wnt polypeptide. In some instances, thefunctional activity of the Wnt polypeptides is detected using assayssuch as for example mass spectroscopy, assays associated with biomarkeranalysis which are described elsewhere herein, transplant surgery suchas sub-renal capsule transplant surgery, spinal fusion surgery, ALP,TRAP, and TUNEL staining, immunohistochemistry, and Micro-CT analysesand quantification of graft growth.

In some instances, the term “stable” refers to Wnt polypeptides as in afolded state and is not unfolded or degraded. In some instances, theterm “stable” also refers to Wnt polypeptides retaining functionalactivity without substantial loss of activity. In some instances, assaysused to determine stability assays that establish the activity of theWnt polypeptides, as such those described above, and also include suchas LSL cell-based assays such as mice LSL cell-based assay.

In some embodiments, the purification method described herein exploitsthe hydrophobic domains of the Wnt polypeptide and does not interferewith Wnt polypeptide conformation or functional activity. In someembodiments, the Wnt polypeptide is Wnt3a polypeptide, Wnt5apolypeptide, or Wnt10b polypeptide. In some embodiments, the Wntpolypeptide is Wnt3a polypeptide. In some cases, the hydrophobic domainof the Wnt3a polypeptide contains a palmitoylation on a serine residuecorresponding to the Serine 209 as set forth in SEQ ID NO:1.

In some embodiments, the affinity of Wnt polypeptide to liposome islower than the Wnt polypeptide binding partners. In some embodiments,the Wnt polypeptide is Wnt3a polypeptide, Wnt5b polypeptide, or Wnt10bpolypeptide. In some embodiments, the Wnt polypeptide is Wnt3apolypeptide. In some embodiments, the affinity of Wnt3a polypeptide toliposome is lower than the Wnt3a polypeptide binding partners. In someembodiments, the Wnt3a polypeptide binding partners include Frizzledprotein, its fragments thereof, LRP6 protein, and its fragments thereof.In some embodiments, the affinity of Wnt3a to liposome is between about4 nM and about 50 nM, and about 4.5 nM and about 20 nM, and about 5 nMand about 15 nM. In some embodiments, the affinity of Wnt3a to liposomeis at least about 5.5 nM, 6 nM, 6.5 nM, 7 nM, 7.5 nM, 8 nM, 8.5 nM, 9nM, 9.5 nM, 10 nM, 11 nM, 12 nM, 13 nM, 14 nM, or more. In someembodiments, the affinity of Wnt3a to liposome is at most about 5.5 nM,6 nM, 6.5 nM, 7 nM, 7.5 nM, 8 nM, 8.5 nM, 9 nM, 9.5 nM, 10 nM, 11 nM, 12nM, 13 nM, 14 nM, or less. In some embodiments, the affinity of Wnt3apolypeptide to its binding partners is between about 0.01 nM to about 4nM, about 0.1 nM to about 4 nM, or about 1 nM to about 4 nM. In someinstances, the affinity of Wnt3a polypeptide to its binding partners isat least about 1.1 nM, 1.3 nM, 1.5 nM, 1.7 nM, 2 nM, 2.3 nM, 2.5 nM, 2.7nM, 3 nM, 3.1 nM, 3.2 nM, 3.3 nM, 3.4 nM, 3.5 nM, 3.6 nM, 3.7 nM, 3.8nM, 3.9 nM, or more. In some instances, the affinity of Wnt3apolypeptide to its binding partners is at most about 1.1 nM, 1.3 nM, 1.5nM, 1.7 nM, 2 nM, 2.3 nM, 2.5 nM, 2.7 nM, 3 nM, 3.1 nM, 3.2 nM, 3.3 nM,3.4 nM, 3.5 nM, 3.6 nM, 3.7 nM, 3.8 nM, 3.9 nM, or less.

Different purification schemes yield different amounts of purifiedpolypeptide. In some embodiments, the Wnt3a polypeptide containing apalmitoylation at an amino acid residue that correspond to Cysteine 77as set forth in SEQ ID NO:1 is the active form. In some embodiments, theWnt3a polypeptide containing a palmitoylation at an amino acid residuethat correspond to Serine 209 as set forth in SEQ ID NO:1 is the activeform. In some embodiments, the Wnt3a polypeptide containing apalmitoylation at an amino acid residue that correspond to Serine 209and a palmitoylation at an amino acid residue that correspond toCysteine 77 as set forth in SEQ ID NO:1 is the active form. In someembodiments, the Wnt3a polypeptide containing a palmitoylation at anamino acid residue that correspond to Serine 209 and a palmitoylation atan amino acid residue that correspond to Cysteine 77 as set forth in SEQID NO:1 is not the active form. In some embodiments, the Wnt3apolypeptide containing a palmitoylation at an amino acid residue thatcorrespond to Cysteine 77 as set forth in SEQ ID NO:1 is not the activeform. In some embodiments, mass spectrometry coupled with additionalassays for detecting or measuring the activity of the Wnt polypeptide(e.g. Wnt3a polypeptide) are used to identify the active specie. In somecases, the additional assays include assays associated with biomarkeranalysis which are described elsewhere herein, transplant surgery suchas sub-renal capsule transplant surgery, spinal fusion surgery, ALP,TRAP, and TUNEL staining, immunohistochemistry, and Micro-CT analysesand quantification of graft growth.

In some aspects, the concentration of the liposomal Wnt3a speciecontaining the Serine 209 palmitoylation is higher in the eluted productthan the Wnt3a specie containing the Cysteine 77 palmitoylation. In someembodiments, the concentration of the two Wnt3a species is defined by avalue, such as a ratio. In some embodiments, the ratio of the Wnt3aSerine 209 specie to the Wnt3a Cysteine 77 specie is between about50:50, about 55:45, about 60:40, about 65:35, about 70:30, about 75:25,about 80:20, about 85:15, about 90:10, about 95:5, about 99:1, or about100:0. In some embodiments, the concentration of the liposomal Wnt3aspecie containing the Serine 209 palmitoylation is high in the elutedproduct than the Wnt3a specie containing the Cysteine 77 and the Serine209 palmitoylations. In some embodiments, the concentration of the twoWnt3a species is defined by a value, such as a ratio. In someembodiments, the ratio of the Wnt3a Serine 209 specie to the Wnt3aCysteine 77/Serine 209 specie is between about 50:50, about 55:45, about60:40, about 65:35, about 70:30, about 75:25, about 80:20, about 85:15,about 90:10, about 95:5, about 99:1, or about 100:0.

In some instances, previous purification schemes, including thosedescribed in the references disclosed herein, yield a mixture of the twospecies (e.g. 1:5 single modified/active:dual modified/inactive). Usingthe purification scheme described herein, in some instances, thepredominant polypeptide specie is in the single modified (at Ser209),active configuration. In some instances, the purification methoddescribed herein provides a Wnt3a composition that containspredominantly an active form of the polypeptide that contains lipidmodification at Ser209. In some instances, the purification methoddescribed herein provides a Wnt3a composition that is not contaminatedby other polypeptide species such as Cys77 Wnt3a specie.

In some embodiments, described herein is a method of preparing aliposomal Wnt polypeptide that comprises the steps of contacting asample comprising Wnt polypeptides to an aqueous solution of liposomes,wherein the phospholipids comprising the liposomes have a phasetransition temperature from about 10° C. to about 25° C. In someembodiments, described herein is a method of preparing a liposomal Wntpolypeptide that comprises the steps of contacting a sample comprisingWnt polypeptides to an aqueous solution of liposomes, wherein thephospholipids comprising the liposome have a tail carbon length ofbetween about 12 carbons and about 14 carbons. In some embodiments, theWnt polypeptide is Wnt3a polypeptide, Wnt5a polypeptide, or Wnt10bpolypeptide. In some embodiments, the Wnt polypeptide is Wnt3apolypeptide.

In some embodiments, disclosed herein is a method of preparing aliposomal Wnt3a polypeptide which comprises the steps of (a) harvestingWnt3a polypeptides from a conditioned media comprising Chinese hamsterovary (CHO) cells; (b) introducing the Wnt3a polypeptides to anion-exchange column immobilized with a sulfonated polyaromatic compound;(c) eluting the Wnt3a polypeptides from the ion-exchange columnutilizing a step gradient; and (d) contacting the Wnt3a polypeptidesfrom step (c) with an aqueous solution of liposomes.

In some embodiments, disclosed herein is a method of preparing aliposomal Wnt5a polypeptide which comprises the steps of (a) harvestingWnt5a polypeptides from a conditioned media comprising Chinese hamsterovary (CHO) cells; (b) introducing the Wnt5a polypeptides to anion-exchange column immobilized with a sulfonated polyaromatic compound;(c) eluting the Wnt5a polypeptides from the ion-exchange columnutilizing a step gradient; and (d) contacting the Wnt5a polypeptidesfrom step (c) with an aqueous solution of liposomes.

In some embodiments, disclosed herein is a method of preparing aliposomal Wnt10b polypeptide which comprises the steps of (a) harvestingWnt10b polypeptides from a conditioned media comprising Chinese hamsterovary (CHO) cells; (b) introducing the Wnt10b polypeptides to anion-exchange column immobilized with a sulfonated polyaromatic compound;(c) eluting the Wnt10b polypeptides from the ion-exchange columnutilizing a step gradient; and (d) contacting the Wnt10b polypeptidesfrom step (c) with an aqueous solution of liposomes.

Functional assays to determine the biological activity of Wntpolypeptides are well known in the art and can include, for example,stabilization of β-catenin, growth promotion of stem cells, quantitationof the amount of Wnt polypeptide present in a non-functional assay, e.g.immunostaining, ELISA, quantitation on Coomasie or silver stained gel,etc., and analysis of the ratio of functionally active Wnt to total Wnt.An exemplary assay for biological activity includes contacting a Wntcomposition with cells, e.g. mouse L cells and then culturing the cellsfor a period of time sufficient to stabilize β-catenin, such as for atleast about 1 hour. The cells are then lysed and the cell lysate isresolved by SDS PAGE. The resolved cell lysate components from the SDSPAGE is subsequently transferred to a nitrocellulose and then probedwith antibodies specific for β-catenin. Other assays for analysis of Wntactivity include C57MG transformation and Xenopus animal cap assays forinduction of target genes. See U.S. Pat. No. 7,335,643, for additionalexemplary assays.

C. Methods of Use

Disclosed herein are methods, processes, compositions, and kits forgenerating bone graft materials for use at a site of bone defect. Alsodisclosed herein are methods, processes, compositions, and kits forenhancing mammalian bone marrow cells. In some instances, the bonedefect refers to a bone injury such as a bone fracture in which graftingmaterials are needed to stimulate and guide reparative growth of naturalbone.

In some embodiments, the method refers to a method of generating bonegraft materials, which comprises the steps of (a) contacting a samplecomprising mammalian bone marrow cells ex-vivo with a liposomal WNTpolypeptide; (b) washing the sample to remove free liposomal WNTpolypeptide; and (c) transplanting the liposomal WNT polypeptide treatedbone graft materials into a site of bone defect. In some embodiments,the Wnt polypeptide is Wnt3a polypeptide, Wnt5a polypeptide, or Wnt10bpolypeptide.

In some embodiments, the Wnt polypeptide is Wnt3a polypeptide. In someembodiments, the method refers to a method of generating bone graftmaterials, which comprises the steps of (a) contacting a samplecomprising mammalian bone marrow cells ex-vivo with a liposomal WNT3apolypeptide; (b) washing the sample to remove free liposomal WNT3apolypeptide; and (c) transplanting the liposomal WNT3a polypeptidetreated bone graft materials into a site of bone defect.

In some embodiments, the Wnt polypeptide is Wnt5a polypeptide. In someembodiments, the method refers to a method of generating bone graftmaterials, which comprises the steps of (a) contacting a samplecomprising mammalian bone marrow cells ex-vivo with a liposomal WNT5apolypeptide; (b) washing the sample to remove free liposomal WNT5apolypeptide; and (c) transplanting the liposomal WNT5a polypeptidetreated bone graft materials into a site of bone defect.

In some embodiments, the Wnt polypeptide is Wnt10b polypeptide. In someembodiments, the method refers to a method of generating bone graftmaterials, which comprises the steps of (a) contacting a samplecomprising mammalian bone marrow cells ex-vivo with a liposomal WNT10bpolypeptide; (b) washing the sample to remove free liposomal WNT10bpolypeptide; and (c) transplanting the liposomal WNT10b polypeptidetreated bone graft materials into a site of bone defect.

In some embodiments, the process refers to a mammalian bone marrowcomposition produced by a process that comprises the step of contactingisolated mammalian bone marrow cells ex-vivo with a liposomal WNTpolypeptide, wherein the contacting time is between about 30 minutes andabout 4 hours. In some embodiments, the Wnt polypeptide is Wnt3apolypeptide, Wnt5a polypeptide, or Wnt10b polypeptide.

In some embodiments, the Wnt polypeptide is Wnt3a polypeptide. In someembodiments, the process refers to a mammalian bone marrow compositionproduced by a process which comprises the step of contacting isolatedmammalian bone marrow cells ex-vivo with a liposomal WNT3a polypeptide,wherein the contacting time is between about 30 minutes and about 4hours.

In some embodiments, the Wnt polypeptide is Wnt5a polypeptide. In someembodiments, the process refers to a mammalian bone marrow compositionproduced by a process which comprises the step of contacting isolatedmammalian bone marrow cells ex-vivo with a liposomal WNT5a polypeptide,wherein the contacting time is between about 30 minutes and about 4hours.

In some embodiments, the Wnt polypeptide is Wnt10b polypeptide. In someembodiments, the process refers to a mammalian bone marrow compositionproduced by a process which comprises the step of contacting isolatedmammalian bone marrow cells ex-vivo with a liposomal WNT10b polypeptide,wherein the contacting time is between about 30 minutes and about 4hours.

In some embodiments, the composition refers to a composition of isolatedenhanced mammalian bone marrow cells wherein the cells have an enhancedexpression level in one or more of the biomarkers selected from thegroup consisting of: Runx2, Osterix, Osteocalcin, Osteopontin, alkalinephosphatase, collagen type I, Axin2, Lef1, and Tcf4, after treatmentwith a liposomal WNT polypeptide. In some embodiments, the biomarkersare selected from Osteocalcin, Osteopontin, Axin2, Lef1, and Tcf4. Insome embodiments, the biomarkers are selected from Osteocalcin, andOsteopontin. In some embodiments, the Wnt polypeptide is Wnt3apolypeptide, Wnt5a polypeptide, or Wnt10b polypeptide.

In some embodiments, the Wnt polypeptide is Wnt3a polypeptide. In someembodiments, the composition refers to a composition of isolatedenhanced mammalian bone marrow cells wherein the cells have an enhancedexpression level in one or more of the biomarkers selected from thegroup consisting of: Runx2, Osterix, Osteocalcin, Osteopontin, alkalinephosphatase, collagen type I, Axin2, Lef1, and Tcf4, after treatmentwith a liposomal WNT3a polypeptide. In some embodiments, the biomarkersare selected from Osteocalcin, Osteopontin, Axin2, Lef1, and Tcf4. Insome embodiments, the biomarkers are selected from Osteocalcin, andOsteopontin.

In some embodiments, the Wnt polypeptide is Wnt5a polypeptide. In someembodiments, the composition refers to a composition of isolatedenhanced mammalian bone marrow cells wherein the cells have an enhancedexpression level in one or more of the biomarkers selected from thegroup consisting of: Runx2, Osterix, Osteocalcin, Osteopontin, alkalinephosphatase, collagen type I, Axin2, Lef1, and Tcf4, after treatmentwith a liposomal WNT5a polypeptide. In some embodiments, the biomarkersare selected from Osteocalcin, Osteopontin, Axin2, Lef1, and Tcf4. Insome embodiments, the biomarkers are selected from Osteocalcin, andOsteopontin.

In some embodiments, the Wnt polypeptide is Wnt10b polypeptide. In someembodiments, the composition refers to a composition of isolatedenhanced mammalian bone marrow cells wherein the cells have an enhancedexpression level in one or more of the biomarkers selected from thegroup consisting of: Runx2, Osterix, Osteocalcin, Osteopontin, alkalinephosphatase, collagen type I, Axin2, Lef1, and Tcf4, after treatmentwith a liposomal WNT10b polypeptide. In some embodiments, the biomarkersare selected from Osteocalcin, Osteopontin, Axin2, Lef1, and Tcf4. Insome embodiments, the biomarkers are selected from Osteocalcin, andOsteopontin.

In some embodiments, the composition also refers to a composition ofmammalian bone marrow cells obtained from a human subject at or olderthan 35 years of age, wherein the mammalian bone marrow cells express anenhanced expression level in one or more of the biomarkers selected fromthe group consisting of: Runx2, Osterix, Osteocalcin, Osteopontin,alkaline phosphatase, collagen type I, Axin2, Lef1, and Tcf4, aftertreatment with a liposomal WNT polypeptide. In some embodiments, thebiomarkers are selected from Osteocalcin, Osteopontin, Axin2, Lef1, andTcf4. In some embodiments, the biomarkers are selected from Osteocalcin,and Osteopontin. In some embodiments, the Wnt polypeptide is Wnt3apolypeptide, Wnt5a polypeptide, or Wnt10b polypeptide.

In some embodiments, the Wnt polypeptide is Wnt3a polypeptide. In someembodiments, the composition also refers to a composition of mammalianbone marrow cells obtained from a human subject at or older than 35years of age, wherein the mammalian bone marrow cells express anenhanced expression level in one or more of the biomarkers selected fromthe group consisting of: Runx2, Osterix, Osteocalcin, Osteopontin,alkaline phosphatase, collagen type I, Axin2, Lef1, and Tcf4, aftertreatment with a liposomal Wnt3a polypeptide. In some embodiments, thebiomarkers are selected from Osteocalcin, Osteopontin, Axin2, Lef1, andTcf4. In some embodiments, the biomarkers are selected from Osteocalcin,and Osteopontin.

In some embodiments, the Wnt polypeptide is Wnt5a polypeptide. In someembodiments, the composition also refers to a composition of mammalianbone marrow cells obtained from a human subject at or older than 35years of age, wherein the mammalian bone marrow cells express anenhanced expression level in one or more of the biomarkers selected fromthe group consisting of: Runx2, Osterix, Osteocalcin, Osteopontin,alkaline phosphatase, collagen type I, Axin2, Lef1, and Tcf4, aftertreatment with a liposomal Wnt5a polypeptide. In some embodiments, thebiomarkers are selected from Osteocalcin, Osteopontin, Axin2, Lef1, andTcf4. In some embodiments, the biomarkers are selected from Osteocalcin,and Osteopontin.

In some embodiments, the Wnt polypeptide is Wnt10b polypeptide. In someembodiments, the composition also refers to a composition of mammalianbone marrow cells obtained from a human subject at or older than 35years of age, wherein the mammalian bone marrow cells express anenhanced expression level in one or more of the biomarkers selected fromthe group consisting of: Runx2, Osterix, Osteocalcin, Osteopontin,alkaline phosphatase, collagen type I, Axin2, Lef1, and Tcf4, aftertreatment with a liposomal Wnt10b polypeptide. In some embodiments, thebiomarkers are selected from Osteocalcin, Osteopontin, Axin2, Lef1, andTcf4. In some embodiments, the biomarkers are selected from Osteocalcin,and Osteopontin.

In some embodiments, the enhanced expression level is compared tountreated mammalian bone marrow cells. In some instances, the enhancedexpression level is between about 1% and about 100%, about 1% and about50%, about 2% and about 20%, about 3% and about 15%, or about 4% andabout 10% compared to untreated mammalian bone marrow cells. In someinstances, the enhanced expression level is at least about 5%, 5.5% 6%,6.5% 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or more compared to untreatedmammalian bone marrow cells. In some instances, the enhanced expressionlevel is at most about 5%, 5.5% 6%, 6.5% 7%, 7.5%, 8%, 8.5%, 9%, 9.5%,or less compared to untreated mammalian bone marrow cells.

In some embodiments, the mammalian bone marrow cells further have adecreased expression level in a biomarker selected from SOX9 and PPARγafter treatment with liposomal WNT polypeptide. In some instances, thedecreased expression level in the mammalian bone marrow cells arecompared to untreated mammalian bone marrow cells. In some cases, thedecreased expression level is between about 1% and about 100%, about 2%and about 80%, about 3% and about 50%, about 3% and about 30%, or about4% and about 20% compared to untreated mammalian bone marrow cells.

In some embodiments, the bone marrow cells further comprise a decreasein apoptosis level compared to untreated mammalian bone marrow cells. Insome instances, the decrease in apoptosis level is at least about 1%,5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, or morecompared to untreated mammalian bone marrow cells. In some instances,the decrease in apoptosis level is at most about 1%, 5%, 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, or less compared to untreatedmammalian bone marrow cells.

In some embodiments, the mammalian bone marrow cells comprise enhancedosteogenic potential compared to untreated mammalian bone marrow cells.In some embodiments, the enhanced osteogenic potential comprises newbone growth level at the site of bone defect after transplanting treatedmammalian bone marrow cells. In some instances, the new bone growthlevel of transplanted treated mammalian bone marrow cells is betweenabout 1% and about 20% or about 5% and about 12% compared to the newbone growth level of transplanted untreated mammalian bone marrow cells.In some instances, the new bone growth level of transplanted treatedmammalian bone marrow cells is increased by about 1.5-fold, about2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, about20-fold, about 50-fold, about 100-fold, or more compared to the new bonegrowth level of transplanted untreated mammalian bone marrow cells.

The bone graft material, as used herein, refers to a cellularcomposition obtained from a donor, in which the donor can be living orcadaveric. Bone graft material typically comprises complex cellpopulations, and includes stem cells such as mesenchymal stem cells, andin some instances, also comprises osteocytes and progenitors thereof. Insome instances, the cells are adherent bone marrow cells or non-adherentbone marrow cells. In some instances, the bone marrow cells are adherentbone marrow cells. In some instances, the adherent bone marrow cells arebone marrow stem cells or bone marrow progenitor cells. In someinstances, the adherent bone marrow cells are bone marrow stromal cells.In some embodiments, the donor is allogeneic. In some instances, thedonor is autologous relative to the recipient. The quantity of cells fora bone graft can vary with the donor, the recipient, purpose of graft,and the like. A bone graft can comprise up to about 10³, up to about10⁴, up to about 10⁵, up to about 10⁶, up to about 10⁷, up to about 10⁸,up to about 10⁹, up to about 10¹⁰ or more cells.

The bone graft material is obtained from the donor, for example from theiliac crest, from the mandibular symphysis (chin area), from reaming,aspirating, and irrigating the femur and/or tibia, fibula, ribs,anterior mandibular ramus; parts of spinal bone, e.g. those removedduring surgery, cadaver bones, etc. In some instances, the graftmaterial is harvested from bone marrow, for example scraped from theendosteal surface of a suitable bone, or from a block graft containingmarrow and a small block of bone. In some cases, allograft bone is takenfrom cadavers, bone banks, etc. for example from a femoral head from hipreplacement surgery. In some instances, the bone graft material isfresh, or is cryo-preserved as known in the art until it is needed.

In some embodiments, the cells of the bone graft are suspended in asuitable culture medium in the presence of an effective dose of aliposomal Wnt polypeptide, e.g. L-Wnt3a, L-Wnt5a, or L-Wnt10b. Anysuitable medium can be used, e.g. DMEM, RPMI, PBS, etc. Cells aretypically resuspended at a concentration that maintains viability duringthe incubation procedure, e.g. up to about 10⁴/ml, up to about 10⁵/ml,up to about 10⁶/ml, up to about 10⁷/ml.

In some instances, the contacting temperature is between about 0° C. andabout 37° C., or about 20° C. and about 25° C. In some embodiments, thecontacting temperature is lower, e.g. up to about 32° C., up to about25° C., up to about 15° C., up to about 10° C., up to about 8° C., up toabout 4° C., up to about 1° C., but typically above freezing unlessspecifically prepared for cryopreservation. In some instances, theincubation temperature is at least about 20° C., 21° C., 22° C., 23° C.,24° C., 25° C., or more. In some instances, the incubation temperatureis at most about 20° C., 21° C., 22° C., 23° C., 24° C., 25° C., orless.

In some cases, the bone graft material contacts with the Wnt polypeptidefor at least about 10 minutes, at least about 30 minutes, at least about1 hour, at least about 2 hours, at least about 3 hours, and up to about36 hours, up to about 24 hours, up to about 18 hours, up to about 15hours, up to about 12 hours, up to about 8 hours, up to about 6 hours,or up to about 4 hours.

The effective dose of the Wnt polypeptide can vary depending on thesource, purity, preparation method, etc. In some instances, theeffective dose of the Wnt polypeptide is at least about 0.01 μg/ml, 0.05μg/ml, 0.1 μg/ml, 0.15 μg/ml, 0.2 μg/ml, 0.25 μg/ml, 0.3 μg/ml, 0.35μg/ml, 0.4 μg/ml, 0.45 μg/ml, 0.5 μg/ml, 0.6 μg/ml, 0.7 μg/ml, 0.8μg/ml, 0.9 μg/ml, 1.0 μg/ml, 1.5 μg/ml, 2.0 μg/ml, 2.5 μg/ml, 3.0 μg/ml,3.5 μg/ml, 4.0 μg/ml, 4.5 μg/ml, 5.0 μg/ml, 7.5 μg/ml, 10 μg/ml, 15μg/ml, 25 μg/ml, 50 μg/ml, 100 μg/ml, or more. In some instances, theeffective dose of the Wnt polypeptide is at most about 0.01 μg/ml, 0.05μg/ml, 0.1 μg/ml, 0.15 μg/ml, 0.2 μg/ml, 0.25 μg/ml, 0.3 μg/ml, 0.35μg/ml, 0.4 μg/ml, 0.45 μg/ml, 0.5 μg/ml, 0.6 μg/ml, 0.7 μg/ml, 0.8μg/ml, 0.9 μg/ml, 1.0 μg/ml, 1.5 μg/ml, 2.0 μg/ml, 2.5 μg/ml, 3.0 μg/ml,3.5 μg/ml, 4.0 μg/ml, 4.5 μg/ml, 5.0 μg/ml, 7.5 μg/ml, 10 μg/ml, 15μg/ml, 25 μg/ml, 50 μg/ml, 100 μg/ml, or less.

In some embodiments, the Wnt polypeptide is L-Wnt3a, the effective doseof the Wnt3a polypeptide is at least about 0.01 μg/ml, 0.05 μg/ml, 0.1μg/ml, 0.15 μg/ml, 0.2 μg/ml, 0.25 μg/ml, 0.3 μg/ml, 0.35 μg/ml, 0.4μg/ml, 0.45 μg/ml, 0.5 μg/ml, 0.6 μg/ml, 0.7 μg/ml, 0.8 μg/ml, 0.9μg/ml, 1.0 μg/ml, 1.5 μg/ml, 2.0 μg/ml, 2.5 μg/ml, 3.0 μg/ml, 3.5 μg/ml,4.0 μg/ml, 4.5 μg/ml, 5.0 μg/ml, 7.5 μg/ml, 10 μg/ml, 15 μg/ml, 25μg/ml, 50 μg/ml, 100 μg/ml, or more. In some instances, the effectivedose of the Wnt3a polypeptide is at most about 0.014/ml, 0.05 μg/ml, 0.1μg/ml, 0.15 μg/ml, 0.2 μg/ml, 0.25 μg/ml, 0.3 μg/ml, 0.35 μg/ml, 0.4μg/ml, 0.45 μg/ml, 0.5 μg/ml, 0.6 μg/ml, 0.7 μg/ml, 0.8 μg/ml, 0.9μg/ml, 1.0 μg/ml, 1.5 μg/ml, 2.0 μg/ml, 2.5 μg/ml, 3.0 μg/ml, 3.5 μg/ml,4.0 μg/ml, 4.5 μg/ml, 5.0 μg/ml, 7.5 μg/ml, 10 μg/ml, 15 μg/ml, 25μg/ml, 50 μg/ml, 100 μg/ml, or less.

In some instances, the Wnt polypeptide is L-Wnt5a, the effective dose ofthe Wnt5a polypeptide is at least about 0.01 μg/ml, 0.05 μg/ml, 0.1μg/ml, 0.15 μg/ml, 0.2 μg/ml, 0.25 μg/ml, 0.3 μg/ml, 0.35 μg/ml, 0.4μg/ml, 0.45 μg/ml, 0.5 μg/ml, 0.6 μg/ml, 0.7 μg/ml, 0.8 μg/ml, 0.9μg/ml, 1.0 μg/ml, 1.5 μg/ml, 2.0 μg/ml, 2.5 μg/ml, 3.0 μg/ml, 3.5 μg/ml,4.0 μg/ml, 4.5 μg/ml, 5.0 μg/ml, 7.5 μg/ml, 10 μg/ml, 15 μg/ml, 25μg/ml, 50 μg/ml, 100 μg/ml, or more. In some instances, the effectivedose of the Wnt5a polypeptide is at most about 0.01 μg/ml, 0.05 μg/ml,0.1 μg/ml, 0.15 μg/ml, 0.2 μg/ml, 0.25 μg/ml, 0.3 μg/ml, 0.35 μg/ml, 0.4μg/ml, 0.45 μg/ml, 0.5 μg/ml, 0.6 μg/ml, 0.7 μg/ml, 0.8 μg/ml, 0.9μg/ml, 1.0 μg/ml, 1.5 μg/ml, 2.0 μg/ml, 2.5 μg/ml, 3.0 μg/ml, 3.5 μg/ml,4.0 μg/ml, 4.5 μg/ml, 5.0 μg/ml, 7.5 μg/ml, 10 μg/ml, 15 μg/ml, 25μg/ml, 50 μg/ml, 100 μg/ml, or less.

In some instances, the Wnt polypeptide is L-Wnt10b, the effective doseof the Wnt10b polypeptide is at least about 0.01 μg/ml, 0.05 μg/ml, 0.1μg/ml, 0.15 μg/ml, 0.2 μg/ml, 0.25 μg/ml, 0.3 μg/ml, 0.35 μg/ml, 0.4μg/ml, 0.45 μg/ml, 0.5 μg/ml, 0.6 μg/ml, 0.7 μg/ml, 0.8 μg/ml, 0.9μg/ml, 1.0 μg/ml, 1.5 μg/ml, 2.0 μg/ml, 2.5 μg/ml, 3.0 μg/ml, 3.5 μg/ml,4.0 μg/ml, 4.5 μg/ml, 5.0 μg/ml, 7.5 μg/ml, 10 μg/ml, 15 μg/ml, 25μg/ml, 50 μg/ml, 100 μg/ml, or more. In some instances, the effectivedose of the Wnt10b polypeptide is at most about 0.01 μg/ml, 0.05 μg/ml,0.1 μg/ml, 0.15 μg/ml, 0.2 μg/ml, 0.25 μg/ml, 0.3 μg/ml, 0.35 μg/ml, 0.4μg/ml, 0.45 μg/ml, 0.5 μg/ml, 0.6 μg/ml, 0.7 μg/ml, 0.8 μg/ml, 0.9μg/ml, 1.0 μg/ml, 1.5 μg/ml, 2.0 μg/ml, 2.5 μg/ml, 3.0 μg/ml, 3.5 μg/ml,4.0 μg/ml, 4.5 μg/ml, 5.0 μg/ml, 7.5 μg/ml, 10 μg/ml, 15 μg/ml, 25μg/ml, 50 μg/ml, 100 μg/ml, or less.

In some embodiments, the bone graft material is incubated with aWntpolypeptide for a period of time sufficient to enhance osteogeniccapacity. The enhancement can be measured in various ways such as theuse of biomarkers described herein, e.g. by increased expression ofAxin2, by increased mitotic activity in the bone graft material(measured at from about day 2 to about day 6 post-transplantation); byincreased bone formation post-transplantation, by increased expressionof Runx2 or Osteocalcin, by reduced apoptosis post-transplantation; orby volume of bone produced post-transplantation.

Following incubation, the bone graft material can be transplanted into arecipient following conventional protocols, e.g. for repair of spinalbone, fractures, dental supports, and the like.

In some embodiments, prior to transplanting the Wnt-treated bone graftmaterial back to the patient, a wash step is performed to remove anyfree liposomal Wnt polypeptides. In some embodiments, at least about 1,2, 3, 4, 5 wash times or more is performed. In some embodiments, at mostabout 1, 2, 3, 4, 5 wash times or less is performed. In someembodiments, any suitable buffer is used for washing, such as forexample phosphate buffered saline solution.

In some instances, the remaining liposomal Wnt after the washing step isat most about 0.01 pg (picogram), 0.05 pg, 0.1 pg, 0.5 pg, 10 pg, 1.5pg, 2 pg, 2.5 pg, 3 pg, 3.5 pg, 4 pg, 4.5 pg, 5 pg, 5.5 pg, 6 pg, orless. In some instances, the remaining liposomal Wnt after the washingstep is at least about 0.01 pg (picogram), 0.05 pg, 0.1 pg, 0.5 pg, 10g, 1.5 pg, 2 pg, 2.5 pg, 3 pg, 3.5 pg, 4 pg, 4.5 pg, 5 pg, 5.5 pg, 6 pg,or more.

In some instances, the method described herein for treatment of bonegraft materials with Wnt allows for control of exposure time of the Wntpolypeptide to cells, duration of the exposure, concentration ofexposure, and/or toxicity. In some instances, the method describedherein for treatment of bone graft materials with Wnt3a allows forcontrol of exposure time of the Wnt3a polypeptide to cells, duration ofthe exposure, concentration of exposure, and/or toxicity.

In some embodiments, osteogenic potential is restored to aged bonegrafts by incubation with a Wnt polypeptide, such as for exampleL-Wnt3a, L-Wnt5a, or L-Wnt10b. In some instances, liposomal Wnt3atreatment reduces cell death in aged bone grafts. In some instancesafter transplantation, bone grafts treated with liposomal Wnt3a gaverise to more bone (p<0.05). In some instances, liposomal Wnt3a treatmentenhanced cell survival in the graft and re-established the bone-formingability of grafts from aged animals.

In some embodiments, mammalian bone marrow cells are obtained from anymammal, including but not limited to a human. In some instances, thedonor is a rodent, such as a mice, or rat. In some instances, the donoris a rabbit. In some instances, the donor is a human. In some instances,the donor is self and the bone marrow cells are autologous. In someinstances, as described elsewhere herein, autologous bone marrow cellsinclude adherent and non-adherent bone marrow cells. In some instances,the autologous bone marrow cells are adherent bone marrow cells. In someinstances, the autologous bone marrow cells include bone marrow stemcells and bone marrow progenitor cells. In some instances, theautologous bone marrow cells include bone marrow stromal cells. In someinstances, the donor is another individual and the bone marrow cells areallogeneic. In some instances, the allogeneic bone marrow cells areobtained from a living donor or a cadaveric donor. In some cases, asdescribed elsewhere herein, the allogenic bone marrow cells includeadherent and non-adherent bone marrow cells. In some cases, theallogeneic bone marrow cells are adherent bone marrow cells. In somecases, the allogeneic bone marrow cells include bone marrow stem cellsand bone marrow progenitor cells. In some cases, the allogeneic bonemarrow cells include bone marrow stromal cells. In some instances, thedonor is an individual at or older than 35 years of age. In someinstances, the bone marrow cells obtained from an individual at or olderthan 35 years of age is considered old bone marrow cells or aged bonemarrow cells. In some embodiments, the donor is an individual youngerthan 35 years of age. In some instances, the bone marrow cells obtainedfrom an individual younger than 35 years of age is considered young bonemarrow cells. In some instances, the bone marrow cells obtained from anindividual at or older than 35 years of age is autologous bone marrowcells or allogeneic bone marrow cells. In some instances, the bonemarrow cells obtained from an individual younger than 35 years of age isautologous bone marrow cells or allogenic bone marrow cells. As usedherein, the terms “individual(s)”, “subject(s)”, “donor(s)”, and“patient(s)” are used interchangeably herein, and means any mammal,including but not limited to a human.

In some embodiments, the methods, compositions, processes, and kitsdescribed herein provide a clinically applicable regenerativemedicine-based strategy for revitalizing bone grafts from aged patients,and from other patients with diminished healing potential, such as, forexample, smokers, diabetics, or patients, with nutritional deficits.

In some embodiments, methods for determining the expression level or thepresence of biomarkers such as Runx2, Osterix, Osteocalcin, Osteopontin,alkaline phosphatase, collagen type I, Axin2, Lef1, Tcf4, SOX9 and PPARγare well known in the art. In some instances, the expression level orthe presence of biomarkers are measured, for example, by flow cytometry,immunohistochemistry, Western Blot, immunoprecipitation, magnetic beadselection, and quantification of cells expressing either of thesebiomarkers. Biomarker RNA or DNA expression levels could be measured byRT-PCR, Qt-PCR, microarray, Northern blot, or other similartechnologies. As used herein, the term “biomarker” and “marker” are usedinterchangeably.

As disclosed herein, determining the expression or presence of thebiomarker of interest at the polypeptide or nucleotide level isaccomplished using any detection method known to those of skill in theart. By “detecting expression” or “detecting the level of” is intendedto determine the expression level or presence of a biomarker protein orgene in the biological sample. Thus, “detecting expression” encompassesinstances where a biomarker is determined not to be expressed, not to bedetectably expressed, expressed at a low level, expressed at a normallevel, or overexpressed.

In certain aspects, the expression or presence of these variousbiomarkers and any clinically useful prognostic markers in a biologicalsample are detected at the protein or nucleic acid level, using, forexample, immunohistochemistry techniques or nucleic acid-basedtechniques such as in situ hybridization and RT-PCR. In someembodiments, the expression or presence of one or more biomarkers iscarried out by a means for nucleic acid amplification, a means fornucleic acid sequencing, a means utilizing a nucleic acid microarray(DNA and RNA), or a means for in situ hybridization using specificallylabeled probes.

In other embodiments, the determining the expression or presence of oneor more biomarkers is carried out through gel electrophoresis. In oneembodiment, the determination is carried out through transfer to amembrane and hybridization with a specific probe.

In other embodiments, the determining the expression or presence of oneor more biomarkers is carried out by a diagnostic imaging technique.

In still other embodiments, the determining the expression or presenceof one or more biomarkers is carried out by a detectable solidsubstrate. In one embodiment, the detectable solid substrate isparamagnetic nanoparticles functionalized with antibodies.

In some embodiments, expression of a biomarker is detected on a proteinlevel using, for example, antibodies that are directed against specificbiomarker proteins. These antibodies are used in various methods such asWestern blot, ELISA, multiplexing technologies, immunoprecipitation, orimmunohistochemistry techniques. In some embodiments, detection ofbiomarkers is accomplished by ELISA. In some embodiments, detection ofbiomarkers is accomplished by electrochemiluminescence (ECL).

In some embodiments, expression level of a biomarker protein of interestin a biological sample is detected by means of a binding protein capableof interacting specifically with that biomarker protein or afunctionally active variant thereof. In some embodiments, labeledantibodies, binding portions thereof, or other binding partners areused. The word “label” when used herein refers to a detectable compoundor composition that is conjugated directly or indirectly to the antibodyso as to generate a “labeled” antibody. In some embodiments, the labelis detectable by itself (e.g., radioisotope labels or fluorescentlabels) or, in the case of an enzymatic label, catalyzes chemicalalteration of a substrate compound or composition that is detectable.

The antibodies for detection of a biomarker protein are eithermonoclonal or polyclonal in origin, or are synthetically orrecombinantly produced. The amount of complexed protein, for example,the amount of biomarker protein associated with the binding protein, forexample, an antibody that specifically binds to the biomarker protein,is determined using standard protein detection methodologies known tothose of skill in the art. A detailed review of immunological assaydesign, theory and protocols are found in numerous texts in the art(see, for example, Ausubel et al., eds. (1995) Current Protocols inMolecular Biology) (Greene Publishing and Wiley-Interscience, NY));Coligan et al., eds. (1994) Current Protocols in Immunology (John Wiley& Sons, Inc., New York, N.Y.).

The choice of marker used to label the antibodies will vary dependingupon the application. However, the choice of the marker is readilydeterminable to one skilled in the art. These labeled antibodies areused in immunoassays as well as in histological applications to detectthe presence of any biomarker or protein of interest. The labeledantibodies are either polyclonal or monoclonal. Further, the antibodiesfor use in detecting a protein of interest are labeled with aradioactive atom, an enzyme, a chromophoric or fluorescent moiety, or acolorimetric tag as described elsewhere herein. The choice of tagginglabel also will depend on the detection limitations desired. Enzymeassays (ELISAs) typically allow detection of a colored product formed byinteraction of the enzyme-tagged complex with an enzyme substrate.Radionuclides that serve as detectable labels include, for example,1-131, 1-123, 1-125, Y-90, Re-188, Re-186, At-211, Cu-67, Bi-212, andPd-109. Examples of enzymes that serve as detectable labels include, butare not limited to, horseradish peroxidase, alkaline phosphatase,beta-galactosidase, and glucose-6-phosphate dehydrogenase. Chromophoricmoieties include, but are not limited to, fluorescein and rhodamine. Theantibodies are conjugated to these labels by methods known in the art.For example, enzymes and chromophoric molecules are conjugated to theantibodies by means of coupling agents, such as dialdehydes,carbodiimides, dimaleimides, and the like. Alternatively, conjugationoccurs through a ligand-receptor pair. Examples of suitableligand-receptor pairs are biotin-avidin or biotin-streptavidin, andantibody-antigen.

As used herein, the term “antibody” is used in the broadest sense andcovers fully assembled antibodies, antibody fragments that can bindantigen (e.g., Fab, F(ab′)₂, Fv, single chain antibodies, diabodies,antibody chimeras, hybrid antibodies, bispecific antibodies, humanizedantibodies, and the like), and recombinant peptides comprising theforgoing.

The terms “monoclonal antibody” and “mAb” as used herein refer to anantibody obtained from a substantially homogeneous population ofantibodies, i.e., the individual antibodies comprising the populationare identical except for possible naturally occurring mutations that canbe present in minor amounts.

Native antibodies” and “native immunoglobulins” are usuallyheterotetrameric glycoproteins of about 150,000 daltons, composed of twoidentical light (L) chains and two identical heavy (H) chains. Eachlight chain is linked to a heavy chain by one covalent disulfide bond,while the number of disulfide linkages varies among the heavy chains ofdifferent immunoglobulin isotypes. Each heavy and light chain also hasregularly spaced intrachain disulfide bridges. Each heavy chain has atone end a variable domain (V_(H)) followed by a number of constantdomains. Each light chain has a variable domain at one end (V_(L)) and aconstant domain at its other end; the constant domain of the light chainis aligned with the first constant domain of the heavy chain, and thelight chain variable domain is aligned with the variable domain of theheavy chain. Particular amino acid residues are believed to form aninterface between the light and heavy-chain variable domains.

The term “variable” refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies.Variable regions confer antigen-binding specificity. However, thevariability is not evenly distributed throughout the variable domains ofantibodies. It is concentrated in three segments called complementaritydetermining regions (CDRs) or hypervariable regions, both in the lightchain and the heavy-chain variable domains. The more highly conservedportions of variable domains are celled in the framework (FR) regions.The variable domains of native heavy and light chains each comprise fourFR regions, largely adopting a β-pleated-sheet configuration, connectedby three CDRs, which form loops connecting, and in some cases formingpart of, the β-pleated-sheet structure. The CDRs in each chain are heldtogether in close proximity by the FR regions and, with the CDRs fromthe other chain, contribute to the formation of the antigen-binding siteof antibodies (see, Kabat et al. (1991) NIH PubL. No. 91-3242, Vol. I,pages 647-669). The constant domains are not involved directly inbinding an antibody to an antigen, but exhibit various effectorfunctions, such as Fc receptor (FcR) binding, participation of theantibody in antibody-dependent cellular toxicity, initiation ofcomplement dependent cytotoxicity, and mast cell degranulation.

The term “hypervariable region,” when used herein, refers to the aminoacid residues of an antibody that are responsible for antigen-binding.The hypervariable region comprises amino acid residues from a“complementarily determining region” or “CDR” (i.e., residues 24-34(L1), 50-56 (L2), and 89-97 (L3) in the light-chain variable domain and31-35 (H1), 50-65 (H2), and 95-102 (H3) in the heavy-chain variabledomain; Kabat et al. (1991) Sequences of Proteins of ImmunologicalInterest, 5th Ed. Public Health Service, National Institute of Health,Bethesda, Md.) and/or those residues from a “hypervariable loop” (i.e.,residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the light-chainvariable domain and (H1), 53-55 (H2), and 96-101 (13) in the heavy chainvariable domain; Clothia and Lesk, (1987) J. Mol. Biol., 196:901-917).“Framework” or “FR” residues are those variable domain residues otherthan the hypervariable region residues, as herein deemed.

“Antibody fragments” comprise a portion of an intact antibody,preferably the antigen-binding or variable region of the intactantibody. Examples of antibody fragments include Fab, Fab, F(ab′)2, andFv fragments; diabodies; linear antibodies (Zapata et al. (1995) ProteinEng. 10:1057-1062); single-chain antibody molecules; and multispecificantibodies formed from antibody fragments. Papain digestion ofantibodies produces two identical antigen-binding fragments, called“Fab” fragments, each with a single antigen-binding site, and a residual“Fc” fragment, whose name reflects its ability to crystallize readily.Pepsin treatment yields an F(ab′)2 fragment that has twoantigen-combining sites and is still capable of cross-linking antigen.

“Fv” is the minimum antibody fragment that contains a complete antigenrecognition and binding site. This region consists of a dimer of oneheavy- and one light-chain variable domain in tight, non-covalentassociation. It is in this configuration that the three CDRs of eachvariable domain interact to define an antigen-binding site on thesurface of the V_(H)−V_(L) dimer. Collectively, the six CDRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chainand the first constant domain (C_(H1)) of the heavy chain. Fab fragmentsdiffer from Fab′ fragments by the addition of a few residues at thecarboxy terminus of the heavy chain C_(H1) domain including one or morecysteines from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear a free thiol group. Fab′ fragments are produced by reducing theF(ab′)2 fragment's heavy chain disulfide bridge. Other chemicalcouplings of antibody fragments are also known.

The “light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa (κ) and lambda (λ), based on the amino acid sequences of theirconstant domains.

Depending on the amino acid sequence of the constant domain of theirheavy chains, immunoglobulins can be assigned to different classes.There are five major classes of human immunoglobulins: IgA, IgD, IgE,IgG, and IgM, and several of these can be further divided intosubclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. Theheavy-chain constant domains that correspond to the different classes ofimmunoglobulins are called alpha, delta, epsilon, gamma, and mu,respectively. The subunit structures and three-dimensionalconfigurations of different classes of immunoglobulins are well known.Different isotypes have different effector functions. For example, humanIgG1 and IgG3 isotypes have ADCC (antibody dependent cell-mediatedcytotoxicity) activity.

In some embodiments, expression or presence of one or more biomarkers orother proteins of interest within a biological sample, for example, atissue sample, is determined by radioimmunoassays or enzyme-linkedimmunoassays (ELISAs), competitive binding enzyme-linked immunoassays,dot blot (see, for example, Promega Protocols and Applications Guide,Promega Corporation (1991), Western blot (see, for example, Sambrook etal. (1989) Molecular Cloning, A Laboratory Manual, Vol. 3, Chapter 18(Cold Spring Harbor Laboratory Press, Plainview, N.Y.), chromatographysuch as high performance liquid chromatography (HPLC), or other assaysknown in the art. Thus, the detection assays involve steps such as, butnot limited to, immunoblotting, immunodiffusion, immunoelectrophoresis,or immunoprecipitation.

In some embodiments, the expression or presence of one or more of thebiomarkers described herein are also determined at the nucleic acidlevel. Nucleic acid-based techniques for assessing expression are wellknown in the art and include, for example, determining the level ofbiomarker mRNA in a biological sample. Many expression detection methodsuse isolated RNA. Any RNA isolation technique that does not selectagainst the isolation of mRNA is utilized for the purification of RNA(see, e.g., Ausubel et al., ed. (1987-1999) Current Protocols inMolecular Biology (John Wiley & Sons, New York). Additionally, largenumbers of tissue samples are readily processed using techniques wellknown to those of skill in the art, such as, for example, thesingle-step RNA isolation process disclosed in U.S. Pat. No. 4,843,155.

Thus, in some embodiments, the detection of a biomarker or other proteinof interest is assayed at the nucleic acid level using nucleic acidprobes. The term “nucleic acid probe” refers to any molecule that iscapable of selectively binding to a specifically intended target nucleicacid molecule, for example, a nucleotide transcript. Probes aresynthesized by one of skill in the art, or derived from appropriatebiological preparations. Probes are specifically designed to be labeled,for example, with a radioactive label, a fluorescent label, an enzyme, achemiluminescent tag, a colorimetric tag, or other labels or tags thatare discussed above or that are known in the art. Examples of moleculesthat are utilized as probes include, but are not limited to, RNA andDNA.

For example, isolated mRNA are used in hybridization or amplificationassays that include, but are not limited to, Southern or Northernanalyses, polymerase chain reaction analyses and probe arrays. Onemethod for the detection of mRNA levels involves contacting the isolatedmRNA with a nucleic acid molecule (probe) that hybridize to the mRNAencoded by the gene being detected. The nucleic acid probe comprises of,for example, a full-length cDNA, or a portion thereof, such as anoligonucleotide of at least 7, 15, 30, 50, 100, 250 or 500 nucleotidesin length and sufficient to specifically hybridize under stringentconditions to an mRNA or genomic DNA encoding a biomarker, biomarkerdescribed herein above. Hybridization of an mRNA with the probeindicates that the biomarker or other target protein of interest isbeing expressed.

In some embodiments, the mRNA is immobilized on a solid surface andcontacted with a probe, for example by running the isolated mRNA on anagarose gel and transferring the mRNA from the gel to a membrane, suchas nitrocellulose. In an alternative embodiment, the probe(s) areimmobilized on a solid surface and the mRNA is contacted with theprobe(s), for example, in a gene chip array. A skilled artisan readilyadapts known mRNA detection methods for use in detecting the level ofmRNA encoding the biomarkers or other proteins of interest.

An alternative method for determining the level of an mRNA of interestin a sample involves the process of nucleic acid amplification, e.g., byRT-PCR (see, for example, U.S. Pat. No. 4,683,202), ligase chainreaction (Barany (1991) Proc. Natl. Acad. Sci. USA 88:189 193),self-sustained sequence replication (Guatelli et al. (1990) Proc. Natl.Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwohet al. (1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase(Lizardi et al. (1988) Bio/Technology 6:1197), rolling circlereplication (U.S. Pat. No. 5,854,033) or any other nucleic acidamplification method, followed by the detection of the amplifiedmolecules using techniques well known to those of skill in the art.These detection schemes are especially useful for the detection ofnucleic acid molecules if such molecules are present in very lownumbers. In particular aspects of the invention, biomarker expression isassessed by quantitative fluorogenic RT-PCR (i.e., the TaqMan0 System).

Expression levels of an RNA of interest are monitored using a membraneblot (such as used in hybridization analysis such as Northern, dot, andthe like), or microwells, sample tubes, gels, beads or fibers (or anysolid support comprising bound nucleic acids). See U.S. Pat. Nos.5,770,722, 5,874,219, 5,744,305, 5,677,195 and 5,445,934, which areincorporated herein by reference in its entirety. The detection ofexpression also comprises using nucleic acid probes in solution.

In some embodiments, microarrays are used to determine expression orpresence of one or more biomarkers. Microarrays are particularly wellsuited for this purpose because of the reproducibility between differentexperiments. DNA microarrays provide one method for the simultaneousmeasurement of the expression levels of large numbers of genes. Eacharray consists of a reproducible pattern of capture probes attached to asolid support. Labeled RNA or DNA is hybridized to complementary probeson the array and then detected by laser scanning Hybridizationintensities for each probe on the array are determined and converted toa quantitative value representing relative gene expression levels. See,U.S. Pat. Nos. 6,040,138, 5,800,992 and 6,020,135, 6,033,860, and6,344,316, which are incorporated herein by reference in its entirety.High-density oligonucleotide arrays are particularly useful fordetermining the gene expression profile for a large number of RNAs in asample.

Techniques for the synthesis of these arrays using mechanical synthesismethods are described in, e.g., U.S. Pat. No. 5,384,261, incorporatedherein by reference in its entirety. In some embodiments, an array isfabricated on a surface of virtually any shape or even a multiplicity ofsurfaces. In some embodiments, an array is a planar array surface. Insome embodiments, arrays include peptides or nucleic acids on beads,gels, polymeric surfaces, fibers such as fiber optics, glass or anyother appropriate substrate, see U.S. Pat. Nos. 5,770,358, 5,789,162,5,708,153, 6,040,193 and 5,800,992, each of which is hereby incorporatedin its entirety. In some embodiments, arrays are packaged in such amanner as to allow for diagnostics or other manipulation of anall-inclusive device.

D. Kits/Article of Manufacture

Disclosed herein, in certain embodiments, are kits and articles ofmanufacture for use with one or more methods, processes, andcompositions described herein. Such kits include a carrier, package, orcontainer that is compartmentalized to receive one or more containerssuch as vials, tubes, and the like, each of the container(s) comprisingone of the separate elements to be used in a method described herein.Suitable containers include, for example, bottles, vials, syringes, andtest tubes. In some embodiments, the containers are formed from avariety of materials such as glass or plastic.

The articles of manufacture provided herein contain packaging materials.Examples of packaging materials include, but are not limited to,bottles, tubes, bags, containers, bottles, and any packaging materialsuitable for a selected formulation and intended mode of administrationand treatment.

For example, the container(s) include L-Wnt polypeptides. Such kitsoptionally include an identifying description or label or instructionsrelating to its use in the methods described herein.

A kit typically includes labels listing contents and/or instructions foruse, and package inserts with instructions for use. A set ofinstructions will also typically be included.

In one embodiment, a label is on or associated with the container. Inone embodiment, a label is on a container when letters, numbers or othercharacters forming the label are attached, molded or etched into thecontainer itself; a label is associated with a container when it ispresent within a receptacle or carrier that also holds the container,e.g., as a package insert. In one embodiment, a label is used toindicate that the contents are to be used for a specific therapeuticapplication. The label also indicates directions for use of thecontents, such as in the methods described herein.

Further details of the invention are provided in the followingnon-limiting Examples.

Example 1 Production and Purification of Human Wnt3a Polypeptide

Human WNT3A is a lipid-modified human stem cell growth factor that iseffective in activating adult stem cells and stimulating theirself-renewal and survival. The native human WNT3A 352 amino acidpolypeptide is post-translationally modified by glycosylation andpalmitoylation. The hydrophobic polypeptide can be difficult to purifyto homogeneity at scalable levels.

As described herein and illustrated in FIG. 1, Human WNT3A is secretedfrom CHO cells and purified using an ion exchange (blue sepharose)column. Following purification, recombinant Human WNT3A is reconstitutedinto lipid vesicles consisting of DMPC and cholesterol. Sucrose densitygradient data demonstrates a physical association between WNT3A and theliposome. Further, the liposomal Human Wnt3a polypeptide (L-WNT3A) canbe stable under physiologic, aqueous conditions. In some instances, theformation of a liposomal structure is not a prerequisite for thisinteraction. In some instances, lipids with random structuralcharacteristics are capable of interacting with Human WNT3A andstabilizing the polypeptide in an aqueous environment.

In some aspects, a L-WNT3A formulation is to be used in aninvestigational new drug (IND) Phase I study to treat bone defects inpatients at high risk for delayed bone healing. In some instances,autologous bone graft material (BGM) is harvested, treated with L-WNT3Aex vivo, then washed and pelleted. In some cases, the resultingmaterial, activated BGM (e.g., BGM^(ACT)), is considered as a drugproduct and is ready for immediate use. In some instances, L-WNT3A willnot be directly administered to the patient but will be used to activatethe autologous cells ex vivo. In some instances, for the initial stagesof the program it is expected that the formulation will meet acceptedU.S. Food and Drug Administration (FDA) criteria (purity, stability,etc.) for a systemically administered liposomal protein formulation.

Production of WNT3A from CHO Cells

Human WNT3A polypeptide was introduced into Chinese hamster ovary celllines and was grown as adherent cells at temperatures ranging from32-37° C. The CHO cells grown in suspension were tested to evaluatewhether the CHO cells in suspension could secrete WNT3A efficiently(FIG. 9).

WNT3A-transformed CHO cells were grown in DMEM plus Glutamax,antibiotics, and serum. The WNT3A plasmid was inducible; and a range ofdoxycycline concentrations was tested (FIG. 10). It was observed thatdoxycycline concentration could modulate the amount of Wnt3a secretionby CHO cells. Cells could be grown for up to 10 days, after which a newaliquot of cells was used.

Fetal bovine serum (FBS) was used for WNT3A secretion from CHO cells(FIG. 11).

Conditioned media (CM) from CHO cell cultures were collected, pooled,and stored at 4° C. up to 2 months without loss in activity (FIG. 12).

Prior to purification, the CM was filtered (0.22 micron filter) and 1%TritonX-100 was added. CM (40 mL) was applied to a blue Sepharose column(1.6 ml) equilibrated in 20 mM TrisCI pH 7.5 and 1% CHAPS. Flow-throughwas collected and the column was washed with 4 column volumes of 20 mMTrisCI pH 7.5, 1% CHAPS, 50 mM KCl. The column was washed again with 4column volumes of 20 mM TrisCI pH 7.5, 1% CHAPS, 100 mM KCl. The columnwas then washed with 3 column volumes of 20 mM TrisCI pH 7.5, 1% CHAPS,150 mM KCl; in which from this wash the human WNT3A was eluted. Thecolumn was washed again with 4 column volumes of 20 mM TrisCI pH 7.5, 1%CHAPS, 250 mM KCl. From a 40 mL CM, the yield was 12 μg of polypeptideat approximately 60% purity. Compared to other purification schemes,this method produced WNT3A with higher purity and higher yield. In somecases, the purity was 70%.

WNT3A Production and Liposome

WNT3A characterization. SDS-PAGE/Western blot. SDS PAGE and Coomassieblue staining analyses of WNT3A purification fractions were collectedand analyzed for WNT3A polypeptide purity by 12% SDS polyacrylamide gelstained with Coomassie brilliant blue and silver nitrate (FIG. 2). Shownin FIG. 2, Purification fractions were collected and analyzed for WNT3Apolypeptide purity by 12% SDS polyacrylamide gel stained with coomassiebrilliant blue and silver nitrate. The estimated purity of WNT3A was70%. Pierce 660 protein assay kit and absorbance at 280 nm were used tomeasure the total protein concentration. In each assay a BSA standardcurve was generated to estimate the total protein concentration. WNT3Apolypeptide concentration was determined using a quantitative Westernblot and a WNT3A standard curve.

Mass spectroscopy. Mass spectrometry analysis verified that WNT3Apolypeptide was lipidated and glycosylated (FIG. 3). The polypeptide wasobserved to be palmitolyated at S209.

Shown in FIG. 3, the method to prepare the WNT3A sample for massspectroscopy was as follows. Polypeptide samples were precipitated using4× volume of pre-chilled acetone at −80° C. The samples were stored at−80° C. overnight followed by ultracentrifugation at 10,000 RPM 4° C.for 10 minutes. The supernatant was removed, and the polypeptide wasreconstitution in solution containing 8M Urea and protease max(Promega). The samples were reduced with 5 mM DTT and incubated at 55°C. for 30 minutes. The samples were then allowed to cool to roomtemperature and then alkylated using acrylamide (15 mM) for 30 minutesat room temperature. Samples were then diluted with 50 mM ammoniumbicarbonate, pH 8.0, to concentrations of <1M urea and 1 μg of trypsinprotease (Promega) was added. The digestion was performed overnight at37° C. The reaction was quenched by the addition of 50% formic acid, andthe peptides were immediately cleaned and concentrated on a C18micro-column (NEST group).

Liquid chromatograph and mass spectrometry (LCMS). Peptides werereconstituted in mobile phase A. A Waters NanoAcquity LC run at 300nL/minute using gradients between 80 and 120 minutes in length was used.The analytical column was packed in house and was made with fused silica(75 uM ID) using PEEKE C18 3 uM matrix approximately 15 cm in length. ALTQ Orbitrap Velos mass spectrometer set in data dependent mode (DDA)was used to perform MSMS fragmentation on the intense precursor ions.CAD, HCD and ETD fragmentation techniques were used.

Database Searching and Analysis. The .RAW files from the massspectrometer were searched against the Human Uniprot database usingSequest. Next a custom database containing the WNT3A sequence wascreated and searched, allowing for an array of variable modifications,not limited to STY phosphorylation, ST hexNac, ST Nac, STCpalmitolyation etc. All Sequest searched files were uploaded intoScaffold (Proteome Software) for interrogation and semi-quantitativeanalysis.

In the case of the positive control (WNT3A protein purchased fromStemRD), approximately 43% coverage was achieved. Sequence coverage andunique peptides are highlighted in yellow (FIG. 3A). The modifiedresidues are shown in green. Palmitolyation modification was detectedonly on the C77 residue (FIG. 3B). In the case of purified WNT3A, ˜51%coverage was achieved. Sequence coverage and unique peptides arehighlighted in yellow (FIG. 3C). The modified residues are shown ingreen. Palmitolyation modification was detected on the S211 residue(FIG. 3C,D) and probable myristoleylation modification was detected onresidues S139 and S181.

L-WNT3A Formulation and Characterization.

Liposomal Wnt3a (L-Wnt3a) polypeptides containing net positive,negative, or neutral charges were manufactured by incubating eitherpositive charged lipids, negatively charged lipids, or neutral chargedlipids at room temperature, with purified Wnt3a for at least about 6 hand up to about 24 h. The lipid to cholesterol ratio was maintained at90:10. Following incubation, L-Wnt3a was separated from CHAPS and otherimpurities by centrifuging at force ranging from 16,000-150,000×g for upto 1 h at 4° C. The L-Wnt3a pellet was then resuspended in sterile1×PBS, overlaid with nitrogen gas and stored at 4° C.

Lipid interactions with WNT3A. When combined with lipids of variouscarbon chain lengths, WNT3A demonstrated a functionally relevantinteraction with uncharged lipids (FIG. 15). These data suggest that itwas primarily a hydrophobic interaction that mediated the associationbetween the WNT3A polypeptide and the liposome (FIG. 4).

TEM characterization of L-WNT3A. TEM was used to visualize the range ofdiameters observed for L-WNT3A (FIG. 4). The appearance and size of theL-WNT3A fall within the size range reported for exosomes e.g., 20-100nM. See also Dhamdhere et al. (2014) PLoS ONE 9(1): e83650, hereinspecifically incorporated by reference in its entirety.

Mouse LSL cell-based assay. Mouse LSL cells were stably transfected witha Wnt-responsive luciferase reporter plasmid pSuperTOPFlash (Addgene)and a constitutive LacZ expression construct pEF/Myc/His/LacZ(Invitrogen) for normalizing beta galactosidase activity to cell number.Human embryonic kidney epithelial (HEK293T) cells were stablytransfected with the above two plasmids. Cells (50000 cells/well,96-well plate) were treated with L-WNT3A in DMEM supplemented with 10%FBS (Gibco) and 1% P/S (Cellgro) at a concentration of 10 uL in 150 uLtotal volume, unless otherwise stated. Included also was a serialdilution of purified WNT3A polypeptide.

Cells were incubated overnight at 37° C., 5% CO₂, then washed, lysedwith Lysis Buffer (Applied Biosystems), and the luciferase andβ-galactosidase expression levels quantified using a dual-light combinedreporter gene assay system (Applied Biosysytems). Bioluminescence wasquantified with triplicate reads on a dual light ready luminometer(Berthold). Activity of WNT3A (ng/uL) and L-WNT3A was defined from astandard curve generated by serial dilutions of WNT3A polypeptide (FIG.5). In experiments involving a time course, WNT3A activity was expressedas percent activity. Percent activity was calculated as follows:

${\% \mspace{14mu} {activity}} = {\frac{( \frac{luc}{lac} ){tn}}{( \frac{luc}{lac} )t\; 0}*100}$

Summary of Stability Data

Based on the LSL reporter cell assay and PAGE-Western blot analyses,conditioned media from fermentation was stable at 4° C. for a minimum of2 months. Based on the LSL reporter cell assay and PAGE-Western blotanalyses, L-WNT3A was stable at 4° C. for >106 days (FIG. 6). Based onthe LSL reporter cell assay and PAGE-Western blot analyses, at 23° C.L-WNT3A had a half-life of >48 hours (blue line, FIG. 7). Compare tonaked WNT3A polypeptide, which lost its activity within 5 min at 23° C.

Based on the LSL reporter cell assay and PAGE-Western blot analyses, at37° C. L-WNT3A had a half-life of 10.5 hours (blue line, FIG. 8). Notethat even if a detergent (CHAPS) was added to stabilize the hydrophobicpolypeptide, WNT3A lost its activity within 5 min (red line, FIG. 8).

Characterization of the WNT Cellular Reporter Assay

The LSL reporter cell line was used to assess activity of Wntpolypeptides and agonists. The following characterizations of L-WNT3Awere made using the following reporter assay. Cells were plated at5.0×10⁴ per assay well and the activity of WNT3A and L-WNT3A werequantified using LSL and HEK293 reporter lines. Under these conditions,WNT3A and L-WNT3A showed similar ability to activate Wnt signaling (FIG.13).

Precision and limit of detection: The assay's sensitivity is R²=0.99. InFIG. 14A and FIG. 14B, WNT was shown to reach an optimal concentrationof 0.1ng/μL. The assay's cutoff points were 0.025 ng/μL and 0.2 ng/μL.The LSL assay was unaffected by deliberate variations in WNT3Aconcentration (FIG. 14C). A range of WNT3A concentrations demonstrated aproportionate luciferase response over time, which indicated thereliability of the assay.

The specificity was demonstrated using the Wnt polypeptide agonistR-spondin2, which synergized with WNT polypeptides to activate the WNTpathway. Assay specificity was also demonstrated using WNTSA, which inthe presence of the Wnt co-receptor Ror2 represses beta catenin mediatedactivation of the pathway. As shown in FIG. 13D, neither reagentdemonstrated activity in the LSL reporter assay.

As shown in FIG. 13(A,B), cells were plated at 5.0×10⁴ per assay welland the WNT3A and L-WNT3A activities were both quantified. Under theseconditions, both WNT3A and L-WNT3A showed similar ability to activateWnt signaling. The assay's sensitivity is R2=0.99. Purified human WNT3Apolypeptide was used as a positive control. (C) The LSL assay wasunaffected by deliberate variations in WNT3A concentration. The datafrom a range of concentrations demonstrated a proportionate luciferaseresponse over time which provided an indication of the reliability ofthe assay. (D) Specificity was demonstrated by addition of Rspo2 in thepresence and absence of WNT3A.

Accuracy: the assay had an intra- and inter-assay coefficient ofvariation of 17.2%.

L-WNT3A Dose Response Curves Using Primary MEFs

LSL and HEK293T cells were engineered to be sensitive to Wnt and Wntagonists and therefore provide little meaningful data on therelationship between dose, drug effect, and clinical response. To moreclosely mimic the in vivo cellular response to a Wnt stimulus, mouseembryonic fibroblasts (MEFs) was assayed using expression of the Wnttarget gene Axin2 as a measure of pathway activity.

In primary cells the linear range of effective concentration was0.025-0.1 ng/μL WNT3A (FIG. 14A). L-WNT3A activity was also assayed inbone marrow-derived stem cells, and found the linear range of effectiveconcentrations ranged from 0.004-0.08 ng/μL (FIG. 14B). Thus, primarycell populations exhibited significantly different sensitivities to aWnt stimulus.

Select lipids can substitute for CHAPS to maintain WNT3A. DMPC andcholesterol lipids could substitute for CHAPS to maintain WNT3Aactivity. Liposomes fabricated with other lipids, such as for exampleMPPC, DPPC, DMPS, DMPG, and DMGE, were inactive (FIG. 15).

Lipid Reconstitution of WNT3A

Many proteins denature at high temperatures, and avoiding suchdenaturation at body temperature is a key to extending the duration of aprotein therapeutic. Liposomal packaging preserved the biologicalactivity of Wnt3a and that this formulation can have efficacy inmultiple bone injury applications. The affinity of Wnt3a for theliposome and the stability of this association were characterized. Thekinetics and dynamics of Wnt pathway activation by L-WNT3A weredemonstrated.

Affinity between WNT3A and liposomes. Sucrose density gradient datademonstrated a physical association between WNT3A polypeptide and thelipids (FIG. 16). The sucrose density gradient in FIG. 16 demonstrated aphysical association between WNT3A and lipids. WNT3A polypeptide (FIG.16A) localized to a high-density fraction and this high-density fractionfurther showed WNT activity (FIG. 16B). Western analyses demonstratedthat the majority of WNT3A polypeptide was localized to thishigh-density fraction (FIG. 16C). The phospholipid assay demonstratedthat this high-density fraction contained the majority of the lipids(orange line, FIG. 16B). Thus when compared with WNT3A alone (whichsegregated in low-density fractions 2-3, FIG. 16D,E) L-WNT3A shifted toa high-density fraction, which demonstrated a physical association andnot as an aggregation between WNT3A and the liposome.

Affinity estimates between Wnt3a and liposomes. The Wnt3a/Frizzledbinding affinity was about 3.6 nM. The Wnt3a/liposome binding affinitywas about 9 nM. In a competition assay between L-Wnt3a and Frizzled,Wnt3a was observed in the liposomal pellet (FIG. 17A). After furtherincubation, Wnt3a dissociated from the liposome pellet andpreferentially associated with Frizzled in the supernatant (FIG. 17B).Thus the affinity of Wnt3a for the liposomal membrane was lower than theaffinity of Wnt3a for Frizzled.

In a second competition assay between liposomes and LRP6, LRP6 wasobserved in the supernatant (FIG. 17C). When LRP6 was incubated in thepresence of L-Wnt3a, the LRP6 protein migrated from the supernatant(FIG. 17C) to the L-Wnt3a pellet (FIG. 17D).

Conclusion: Based on these pull-down assays the binding affinity betweenWnt3a and the liposomal membrane was estimated to be about 6 nM. Theseassays also demonstrated that Wnt3a reconstituted in a liposomalmembrane was positioned in such a way that it could interact with itsreceptors Frizzled and LRP6.

Kinetics of WNT3A Association with Liposomes

Based on the LSL reporter cell assay and PAGE-Western blot analyses,Wnt3a rapidly associated with the liposome and this association wasresponsible for maintaining the polypeptide's activity. Initially, Wnt3aactivity was observed in the supernatant. Over the course of about 30minutes, Wnt3a polypeptide transferred from the supernatant to thepellet at a rate of about 3×10⁻³ nM/sec (FIG. 18A). By 6 h, 90% of theWnt3a activity was observed in the pellet. Similarly, western blotanalysis showed that 90% of the polypeptide were found in the pellet(FIG. 18B). After 24 h at 23° C., liposomes were separated from theaqueous supernatant by centrifugation. No visible polypeptideprecipitation was observed in the pellet. Western blot demonstrated thatthe majority of Wnt3a was in the liposomal pellet (FIG. 18C). FIG. 18Dshows the Wnt3a activity in the supernatant and the pellet at the end ofthe experiment.

FIG. 19 illustrates Wnt5a and Wnt10b interactions with liposomes (FIG.19).

Scale up experiment. Cells from the freezer stock were seeded onto a 15cm tissue culture plate. After incubation at 37° C., 5% CO₂ for 3-4 daysthe cells were expanded 1:5 into 2×15 cm plates for 4 days. These cellswere further expanded 1:5 into 20×15 cm plates. After 24 hours ofincubation the cells were induced with doxycycline. CM was collectedevery 24 hours and stored at 4° C. Activity of the CM was measured toconfirm WNT3A secretion. 1% TritonX was added to 1 L CM and filteredthrough a 0.22 μm filter. CM was then loaded onto a 150 ml bluesepharose column. From this trial 80 μg of WNT3A was eluted in agradient of KCl, resulting in 40% overall yield.

Materials and Suppliers

CHO cells: InvitrogenPlasmid vector: Promega

Lipids: Avanti Polar Lipids

Blue sepharose purification column:WNT3A standard: Stem R&DLSL cells:

Ultracentrifuge: Abbreviations

BGM: bone graft materialBGM^(ACT): L-WNT3A activated bone graft material

CHO: Chinese Hamster Ovary

CM: Conditioned mediaFz: Frizzled, the WNT receptorHEK: Human embryonic kidneyLSL: mouse L cellsL-WNT3A: liposomal WNT3AMEF: Mouse embryonic fibroblast

Example 2

Human Wnt3a polypeptide was produced by transfection of a geneticallyengineered Chinese hamster ovary (CHO) cell line with a plasmid carryinghuman Wnt3a nucleic acid. A lipofectin-based transfection method wasused. The plasmid was under the control of a doxycycline-induciblepromoter. The Wnt3a-transformed CHO cells were grown in adherent orsuspension culture in a culture medium containing DMEM (Dulbecco'sModified Eagle Medium)+ 10% fetal bovine serum (FBS), GlutaMax™ (aglutamine-based dipeptide), antibiotic (e.g. doxycycline), G418,non-essential amino acids, blasticidine and serum, and at temperaturesranging from about 32-37° C. The amount of Wnt3a secreted from CHO cellsdepended on whether they were grown as adherent or suspended cultures.Doxycycline was added to the media at a range of concentrations, whichcould modulate the amount of human Wnt3a produced by CHO cells. Theculture medium included Fetal bovine serum (FBS) which was presentthroughout the culture period during which the CHO cells secreted Wnt3a.Cells can be grown for up to 10 days, after which a new aliquot of cellswas used.

Compared to a purification scheme using either a continuous gradient ora gel filtration, the present purification scheme uses a step gradient,which produces higher purity and higher yield as shown in Tables 1 and2. In the purification scheme that uses either a continuous gradient ora gel filtration, the continuous salt gradient was run on a blueSepharose column from 150 mM to 1.5 M NaCl, followed by gel filtrationand a heparin sulfate column. In some instances, as described elsewhereherein, the present scheme also utilizes a hydrophobic purification stepsuch as a hydrophobic column (e.g. a Protein A-Sepharose column).

Table 1 shows results obtained by the purification step of the presentinvention and Table 2 shows results obtained by the purification schemeusing either a continuous gradient or a gel filtration purificationstep.

TABLE 1 Protein Total Wnt3a conc protein protein Fold % Fraction Volume(ug/ml) (mg) (ug) purification Yield Conditioned 40 ml 3714 148.560 20100 media Anion  6 ml 64 0.384 12 60 exchange (Blue Sepharose)

TABLE 2 Protein Total Wnt3a Fold conc protein protein puri- FractionVolume (ug/ml) (mg) (ug) fication % Yield Conditioned 350 ml 37141,299.900 175 — 100 media Anion 120 ml 54 6.480 60 34.2 exchange (BlueSepharose) Gel filtration  30 ml 70.6 2.118 7.5 4.2 (Superdex 200)

Example 3 Animals

All procedures followed protocols approved by the Stanford Committee onAnimal Research. Beta-actin-enhanced green fluorescent protein(ACTB-eGFP; The Jackson Laboratory, Sacramento, Calif.) and CD1 wildtype, syngeneic mice were used. Mice <3 months old were consideredyoung; mice >10 months were considered aged. Axin2^(CreERT/+) andR26R^(mTmG/+) mice were purchased from Jackson Labs. Aged wild typeLewis rats (“retired breeders” from Charles Rivers, Mass.), wereutilized for spinal fusion surgeries according to AAUC and IUPACguidelines.

Collection and Treatment of Bone Graft Material (BGM) for Rodent Models

Both rats and mice were employed in this study. The use of mouse modelsallows for a broad spectrum of molecular analyses, however, becauseautografts are highly invasive for these small animals, rats were usedwhen performing autografts (e.g., FIGS. 20 and 25), and syngeneic micewhen employing advanced molecular techniques (FIGS. 21-24). In allcases, bone graft material (BGM) was harvested from femurs, tibiae andiliac crest by splitting the bones lengthwise, gently scraping theendosteal surface with a sharp instrument, and irrigating the marrowcontents into a collection dish. This method mimicked thereamer/irrigator/aspirator (RIA) technique used in humans.

To induce recombination in Axin2^(CreERT/+); R26R^(mTmG/+) mice (FIG.21), animals were given 160 pg/g body weight tamoxifen via IP injectionor gavage for 5 consecutive days. Tissues were harvested 7 days afterthe first treatment and analyzed by GFP immunostaining or fluorescence.

To ensure that BGM aliquots for transplantation into the sub-renalcapsule (SRC) were equivalent in terms of cellular content, BGM from 3mice (littermates) was pooled then divided into 20 μL aliquots just asin the transplant assays. DNA content was extracted with the DNeasyTissue Kit (QIAGEN) and relative DNA concentration was measured usingthe Quant-iT PicoGreen dsDNA Kit (Invitrogen) and microplatefluorescence reader (BERTHOLD, Bad Wildbad, Germany). The percentvariation in DNA content was <20% (FIG. 26).

To obtain BGM^(ACT), freshly harvested BGM was placed into 20 μL ofculture medium containing a liposomal formulation of eitherphosphate-buffered saline (L-PBS) or WNT3A (L-WNT3A, effective L-WNT3Aconcentration=0.15 μg/mL) and maintained at 23° C. for 1 hour.

Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR)

Tissue samples were homogenized in TRIzol solution. RNA was isolated andreverse transcription was performed. Quantitative real-time PCR wascarried out using Prism 7900HT Sequence Detection System and Power SYBRGreen PCR Master Mix (Applied Biosystems). Levels of gene expressionwere determined by the ΔΔCT method and normalizing to their GAPDHvalues. All reactions were performed in triplicate, means and standarddeviations were calculated.

Primers sequences (5′ to 3′) are as follows: Axin2,[for-TCATTTTCCGAGAACCCACCGC], [rev-GCTCCAGTTTCAGTTTCTCCAGCC]; Lef1,[for-AGGAGCCCAAAAGACCTCAT], [rev-CGTGCACTCAGCTATGACAT]; GAPDH,[for-ACCCAGAAGACTGTGGATGG [rev-GGATGCAGGGATGATGTTCT]; ALP[for-ACCTTGACTGTGGTTACTGC, [rev-CATATAGGATGGCCGTGAAGG]; Osterix,[for-GGAGACCTTGCTCGTAGATTTC], [rev-GGGATCTTAGTGACTGCCTAAC]; Osteocalcin,[for-TGTGACGAGCTATCAAACCAG], [rev-GAGGATCAAGTTCTGGAGAGC].

Western Analyses

BGM was harvested from young (N=5) and aged (N=5) mice and then placedin Dulbecco's Modified Eagle Medium (DMEM) containing 10% Fetal BovineSerum (FBS), 100 U/mL penicillin and 100 pg/mL streptomycin, andincubated at 37° C. in 5% CO₂. After 24 hours, non-adherent cells wereremoved, media was replaced, and adherent cells were passaged until theyreached confluence. Media was changed every 3 days. In some experiments,cells after passage 3 were treated with either L-PBS or L-WNT3A(effective concentration=0.03 μg/mL). In these experiments, cells werecollected 24 h later and lysed using RIPA buffer. Total protein wasextracted for Western analysis. Pan-actin was used as an internalcontrol and to ensure protein integrity. Antibodies against WNT3A,non-phosphorylated β-catenin, and Axin2 were used. Integrated intensitywas analyzed by ImageJ to quantify Western blotting results.

Sub-Renal Capsule Transplant Surgery

In some cases, the sub-renal capsular assay (SRCA) was employed to assayits differentiation potential. Following inhalation of anesthesia by thesyngeneic host mice, a skin incision was made on the left flank directlycaudal to the rib cage. The peritoneal cavity was opened to expose thekidney. A small incision was created in the renal capsule and the BGMswere carefully placed under the capsule using soft plastic tubing. Thekidney was then returned to the peritoneal cavity, and the peritoneumand skin were closed with sutures. Buprenorphine (0.05 mg/kg) was usedfor analgesia.

In cases where the BGM was harvested from Axin2^(CreERT2); R26^(mTmG)donors, hosts were subsequently provided tamoxifen by gavage (100 μL of10 mg/mL) beginning on day 0 for 5 days. The SRC transplants wereharvested at the time points indicated.

Adenovirus-Mediated Inhibition of Wnt Signaling

DKK1 and the negative control Fc adenoviral constructs were generated.The adenoviral constructs were transfected into 293T cells. After 2days, cells were collected, lysed, and precipitated by centrifugation.The purified adenovirus was aliquoted and stored at −80° C. Wntinhibition was achieved by in vitro incubation of BGMs with Ad-Dkk1 andthe control Ad-Fc for 2 hours, and the BGM aliquots were thentransplanted into calvarial defects.

Calvarial Critical-Size Defect Surgery

Mice were anesthetized, and a 3-mm incision was made to expose theparietal bone. A circumferential, full-thickness defect with a 2-mmdiameter was created with the use of a micro-dissecting trephine; thedura mater was not disturbed. BGM aliquots were incubated with Ad-Dkk1and the control Ad-Fc for 2 hours. BGM aliquots were then transplantedinto the calvarial defect and the skin was closed with sutures.Following recovery from surgery, mice received Buprenorphine foranalgesia.

Micro-computed tomography (Micro-CT) analyses were performed as follows.Mice were anesthetized with 2% isoflurane and scanned with use of amultimodal positron emission tomography-computed tomographydata-acquisition system (Inveon PET-CT) at 40-μm resolution. Data wereanalyzed with MicroView software. The three-dimensionalregion-of-interest tool was used to assign the structure and bone volumefor each sample.

Assessment of the regenerate bone volume fraction (the percentagecalculated by dividing the total bone volume by the regenerate bonevolume [BV/TV, %]) was performed with the use of high-resolutionmicro-CT (vivaCT 40), with 70 kVp, 55 μA, 200-ms integration time, and a10.5-μm isotropic voxel size. The region of interest was 2 cm in lengthand began 250 μm proximal to the edge of the defect and extended 250 μmdistally beyond the opposing edge of the defect (1.5 cm total diameter).Bone was segmented from soft tissue with use of a threshold of 275mg/cm3 hydroxyapatite. Scanning and analyses adhered to publishedguidelines.

Spinal Fusion Surgery

Lewis Rats were anesthetized using a cocktail of Ketamine 70-100 mg/kgand Xylazine 5-10 mg/kg. The lumbar region of the rats were shaved thendisinfected with Betadine-soaked gauze. Prior to the skin incision, therats were injected with the analgesia buprenorphine 0.02 mg/kg SC/IP.First, bone graft material (BGM) was harvested from the iliac crest;briefly, the left iliac spine was palpated and a vertical cutaneousincision was made; the dorsal crest of the iliac spine was accessed andexposed through blunt dissection. The attached muscle and periosteumwere elevated and 0.3 g of BGM was harvested with rongeur forceps andmorselized. BGM was then incubated with either 100 μL L-PBS or with 100μL of [0.15 μg/mL] L-Wnt3a while the transverse processes were exposed.

To expose the transverse processes, posterolateral blunt dissection wasperformed and the reflected paraspinal muscles were held in place byretractors. The transverse processes of L4-L5 were cleaned of periosteumand decorticated with a bur. The BGM was spread over and between theL4-L5 transverse processes. The paraspinal muscles were closed withabsorbable sutures (4-0 Vicryl, Ethicon) and the skin with interruptednon-absorbable sutures (4-0 Nylon, Ethicon). The surgical site wastreated with an antibiotic ointment. 10 mg/kg Baytril was deliveredsubcutaneously. Buprenorphine (0.02 mg/kg) was administered aftersurgery for 3 days, and subsequent doses were given as needed to controlpain.

Sample Preparation, Tissue Processing, Histology

Tissues were fixed in 4% paraformaldehyde (PFA) overnight at 4° C.Samples were decalcified in 19% EDTA for 1 day. Specimens weredehydrated through an ascending ethanol series prior to paraffinembedding. Eight-micron-thick longitudinal sections were cut andcollected on Superfrost-plus slides for histology. Safranin 0, Anilineblue and Gomori staining were performed. Tissue sections werephotographed using a Leica DM5000B digital imaging system.

ALP, TRAP and TUNEL Staining

Alkaline phosphatase (ALP) activity was detected by incubation in nitroblue tetrazolium chloride (NBT), 5-bromo-4-chloro-3-indolyl phosphate(BCIP), and NTM buffer (100 mM NaCl, 100 mM Tris pH9.5, 5 mM MgCl).Tartrate-resistant acid phosphatase (TRAP) activity was observed using aLeukocyte acid phosphatase staining kit following manufacturer'sinstructions. After developing, slides were dehydrated in a series ofethanol, cleaned in Citrisolv, and cover-slipped with Permount mountingmedia. For TUNEL staining, sections were permeabilized using 0.1% TritonX-100 and 0.1% sodium citrate, and incubated with TUNEL reaction mixture(In Situ Cell Death Detection Kit). Sections were mounted with DAPImounting medium and visualized under an epifluorescence microscope.

For bromodeoxyuridine (BrdU) assay, mice were given intraperitonealinjections of BrdU labeling reagent (Invitrogen, CA, USA) and euthanized4 hours post-injection. BrdU detection was carried out using BrdUStaining Kit following the manufacturer's instructions.

Immunohistochemistry

Tissue sections were deparaffinized and rehydrated in PBS. Endogenousperoxidase activity was quenched by 3% hydrogen peroxide for 5 min, andthen washed in PBS. Slides were blocked with 5% goat serum for 1 hour atroom temperature. The appropriate primary antibody was added andincubated overnight at 4° C. Samples were then incubated withappropriate biotinylated secondary antibodies and advidin/biotinylatedenzyme complex and developed by a DAB substrate kit. Antibodies usedincluded GFP and DLK1, Runx2, Sox9 and PPAR-γ.

Micro-CT Analyses and Quantification of Graft Growth

Scanning and analyses adhered to published guidelines. Rats wereanesthetized with 2% isoflurane and scanned with a micro computedtomography data-acquisition system (Inveon) at a 52-μm resolution. Todefine the graft growth that occurred in each sample, POD2 and POD49timepoint scanning data were exported into Osirix software version 5.8and registered for segmentation in the same orientation. The new bonethat formed was compared to the initial BGM volume transplanted.Differences between sets of data were determined by using Mann-Whitneytest in XLStat software version. A p-value <0.05 was consideredstatistically significant.

Quantification and Statistical Analyses

GFP, BrdU, TUNEL, DLK1, Osteocalcin and Aniline blue stainings werequantified. Photoshop CS5 was used to determine the number of pixels inthe region of interest (ROI), at the injury site. The magic wand toolwas used to assign the area of positive pixels within the ROI. The ratioof pixels of positive signals to pixels of ROI was expressed as apercentage. At least 5 sections evenly spaced across the injury siteswere quantified to determine the average value of each sample. Fivesamples were included in each group (n=5). Results are presented as themean±SD. Student's t-test was used to quantify differences describedherein. P≤0.05 was considered significant.

Bone Graft Material Contains Multiple Stem/Progenitor Cell Populations

In some cases, the optimal anatomical site for harvesting autograftsdepends upon a number of factors including donor site morbidity and theavailability of bone stock. The BGM was harvested from three anatomicalsites using a modified reamer-irrigator-aspirator (RIA) technique andnoted that the femur, iliac crest, and tibia yielded BGM with distinctlydifferent histological appearances. In addition to hematopoietic cells,femur BGM contained adipocytes, even when harvested from young animals(FIG. 20A). Iliac crest BGM was largely comprised of trabecular bonefragments covered in tightly adherent cells (FIG. 20B). BGM from thetibia contained a considerable amount of fibrous stroma and small,anucleated cells (FIG. 20C). A quantitative RT-PCR was used to evaluateendogenous osteogenic gene expression and found that of the threesources, iliac crest BGM expressed alkaline phosphatase and Osteocalcinat significantly higher levels (FIG. 20D).

In some cases, it is believed that the osteogenic property of anautograft is attributable to stem/progenitor cell populations andosteoblasts within the bone graft material (BGM). This osteogenicproperty of an autograft was tested by transplanting BGM into asub-renal capsule (SRC) assay. The SRC provides a vascular supply to thetransplanted tissue and supports the differentiation of cells intomultiple kinds of tissues including bone, skin, muscle, teeth, organs,and tumors. BGM was harvested from the iliac crest, then transplantedbeneath the animal's kidney capsule (FIG. 20A) and allowed to developthere for 7 days.

BrdU incorporation demonstrated the high mitotic activity of cells inthe autologous BGM (FIG. 20B). Immunostaining for Runx2 (FIG. 20C), Sox9(FIG. 20D) and PPARγ (FIG. 20E) demonstrated that subsets of cells inthe BGM expressed gene markers associated with osteogenic, chondrogenicand adipogenic commitment. On day 7, a sub-population of BGM-derivedcells had differentiated into bone (FIG. 20F), cartilage (FIG. 20G), andfat (FIG. 20H). Together, these data demonstrated that the BGM containedstem/progenitor cells capable of differentiating into all threelineages.

Wnt Signaling in Bone Graft Material Declines with Age

Axin2^(CreERT2); R26^(mTmG) reporter mice were used to inducerecombination which lead to the identification of GFP^(+ve)pre-osteoblasts in the periosteum (FIG. 21A) and the endosteum (FIG.21B). The frequency of GFP^(+ve) cells in the endosteum was ˜0.1% (FIG.21C). GFP^(+ve) cells were also identified in freshly harvested BGM(FIG. 21D). Thus, a subset of cells in the heterogeneous BGM is Wntresponsive.

The Wnt responsive status of BGM was compared between young (<3 monthold) and aged (>10 month old) mice. Quantitative absolute RT-PCRdemonstrated that expression of the Wnt target genes Axin2 and Lef1 wasalmost two-fold lower in BGM harvested from aged mice (BGM^(aged)) v.young mice (BGM^(youg); FIG. 21F). Western analysis confirmed thatWnt3a, phosphorylated beta catenin, and Axin2 expression were allsignificantly lower in BGM^(aged) compared to BGM^(young) (FIG. 21G).Thus, the endogenous Wnt responsive status of BGM deteriorates with age.

Osteogenic Differentiation Capacity Also Declines with Age

In humans, the rate of bone healing declines with age. A similarage-related decline was also found in the osteogenic capacity of BGM inmice. Freshly harvested BGM^(aged) showed significantly lower expressionlevels of the osteogenic genes Alkaline phosphatase, Osterix, andOsteocalcin compared to freshly harvested BGM^(young) (FIG. 22A).

To test whether the reduction in osteogenic gene expression affected theosteogenic capacity of the BGM, the SRC assay was used. Performing anautograft in a mouse, however, is excessively traumatic. To mimic anautograft, syngeneic donors and hosts were used. Because syngeneicanimals are so closely related, their tissues are immunologicallycompatible and transplantation of tissues does not provoke an immuneresponse. ACTB-eGFP mice served as the donors and BGM was readilyidentifiable in the SRC by its GFP fluorescence (FIG. 22B, FIG. 22C).

Seven days after transplantation, BGM was harvested and analyzed forevidence of bone formation. Aniline blue stained osteoid matrix wasevident in BGM^(young) (FIG. 22D) but absent in BGM^(aged) (FIG. 22E;quantified in FIG. 22F). The osteoid matrix in BGM^(young) wasundergoing mineralization as shown by ALP staining (FIG. 22G) whereasBGM^(aged) showed no ALP staining (FIG. 22H; quantified in I). Further,the GFP immunostaining demonstrated that in both BGM^(young) (FIG. 22J)and BGM^(aged) (FIG. 22K) there were a similar number of surviving donorcells (quantified in FIG. 22L). Together these data indicated thatosteogenic gene expression and osteogenic capacity of BGM declined withage.

Wnt Signaling and the Osteogenic Capacity of BGM

Endogenous Wnt responsiveness and the osteogenic capacity of BGMdiminish with age. To test whether reduced Wnt signaling contributed tothis age-related decline in osteogenic potential, endogenous Wntsignaling in BGM was blocked. Over-expression of the Wnt inhibitor,Dkk1, was also used to transiently abolish Wnt signaling in vivo. EitherAd-Dkk1 or Ad-Fc (control) was delivered to the bone marrow cavity ofyoung mice then harvested BGM^(young) 24 h later and transplanted thealiquots into critical size (non-healing) skeletal defects.

Seven days later, when control BGM^(young) was strongly positive for ALPactivity (FIG. 23A), Ad-Dkk1 treated BGM^(young) showed minimal activity(FIG. 23B). Instead, Ad-Dkk1 treated BGM^(young) showed widespreadexpression of the adipogenic proteins PPAR-γ (FIG. 23C, FIG. 23D) andDIM (FIG. 23E, FIG. 23F). Bone formation was repressed by Ad-Dkk1treatment, as shown by micro-CT (FIG. 23G, FIG. 23H; quantified in FIG.23I) and histomorphometric analyses of the BGM (FIG. 23J, FIG. 23K;quantified in FIG. 23L). Thus, the osteogenic capacity of BGM reliesupon an endogenous Wnt signal.

Augmenting the Endogenous Wnt Signal in BGM^(aged) Restores itsOsteogenic Capacity

FIG. 23J-FIG. 23L showed that endogenous Wnt signaling could activateosteogenic capacity of BGM. A Wnt stimulus was also tested forsufficiency to enhance BGM efficacy. BGM^(aged) was harvested, treatedwith L-WNT3A or liposomal PBS (L-PBS), and then incubated at 37° C.(FIG. 24A). Absolute qRT-PCR analyses revealed a small elevation inAxin2 expression (FIG. 24B). Left was modestly elevated in response toL-WNT3A (FIG. 24B). Western analyses indicated that both beta cateninand Axin2 proteins were elevated in response to L-WNT3A (FIG. 24C).

Mitotic activity in BGM was increased by L-WNT3A treatment. Onpost-transplant day 4, cell proliferation was increased in L-WNT3Atreated BGM^(aged) compared to L-PBS-treated BGM^(aged) (FIG. 24D, FIG.24E; quantified in FIG. 24F). The effect on cell cycling was transientby post-transplant day 7, BrdU incorporation was equivalent between theL-PBS and L-WNT3A samples (FIG. 24G, FIG. 24H; quantified in FIG. 24I).

Cell differentiation in BGM^(aged) was evaluated. Expression of theadipogenic protein Dlk1 was lower (FIG. 24J, FIG. 24K; quantified inFIG. 24L) and expression of the osteogenic protein Osteocalcin washigher in L-WNT3A treated BGM^(aged) (FIG. 24M, FIG. 24N; quantified inFIG. 24O). New bone formation was found only in L-WNT3A treatedBGM^(aged) (FIG. 24P, FIG. 24Q; quantified in FIG. 24R). Treatment withL-WNT3A did not affect engraftment efficiency (FIG. 27A,B; quantified inFIG. 27C) but analyses of programmed cell death demonstrated thatL-WNT3A treated BGM^(aged) had fewer TUNEL^(+ve) cells than L-PBStreated samples (FIG. 27D, FIG. 27E; quantified in FIG. 27F). Reducedapoptosis was also observed at post-transplant day 7 (FIG. 27G, FIG.27H; quantified in FIG. 27I). New bone formation served as a stimulusfor osteoclast-mediated bone remodeling and TRAP activity was observedaround the newly formed osteoid matrix in L-WNT3A treated BGM^(aged)samples (FIG. 27J, FIG. 27K; quantified in FIG. 27L).

L-WNT3A Activates Stem Cells in BGM^(aged) and Improves Bone Generationin a Spinal Fusion Model

The population of cells in BGM responsible for the L-WNT3A mediatedsurge in osteogenic capacity was also identified. Two stem cellpopulations from BGM using standard procedures were isolated and wereevaluated based on their Wnt responsiveness using L-PBS (as control) orL-WNT3A.

Time-course analyses revealed the response of stem cells to L-WNT3Atreatment. Within 15 h of L-WNT3A exposure, a 4-fold activation in Axin2was observed, and maximal Axin2 activation was achieved at 36 h (FIG.25A). The effect was transient, as shown by diminished Axin2 expressionin stem cells at the 60 h timepoint (FIG. 25A). A second stem cellpopulation isolated from BGM was used to verify that stem cellsresponded to L-WNT3A (FIG. 25B).

The activated state of the BGM was shown by qRT-PCR. BGM^(aged) washarvested, treated with L-WNT3A or L-PBS, then analyzed using Axin2 andLef1 expression for its Wnt responsive status (FIG. 25C). Within 12h anelevation in Lef1 was detectable. Within 24h, both Axin2 and Lef1 wereelevated (FIG. 25C). These analyses confirm a WNT-mediated activationstate of BGM; hereafter refers to this material as BGM^(ACT).

The therapeutic potential of BGM^(ACT) in a rat spinal fusion model wasalso tested. The transverse processes of the fourth and fifth lumbar(e.g., L4-5) vertebrae were decorticated (FIG. 25D) and during thisprocedure, autologous BGM^(aged) from the iliac crest was harvested andtreated with L-WNT3A (or L-PBS) for 1h. The resulting material,BGM^(ACT) (or BGM^(aged)) was then transplanted onto and between theL4-5 processes (FIG. 25E). On post-operation day 2 the volume of the BGMwas evaluated by micro-CT. These analyses verified that BGM^(aged) (FIG.25F) and BGM^(ACT) (FIG. 25G) contained comparable amounts ofmineralized tissue at the outset (see also FIG. 26).

The volume of new bone formation was re-evaluated on post-operation day49. Three-dimensional reconstructions of the micro-CT data demonstratedpoor bone regeneration in sites treated with BGM^(aged) (grey; FIG.25H). In contrast, sites treated with BGM^(ACT) showed evidence ofrobust bone formation and fusion of the transverse processes (blue; FIG.25I). The volume of new bone on and between the L4 and L5 transverseprocesses was quantified. Compared to BGM^(aged) BGM^(ACT) gave rise tomore mineralized matrix (FIG. 25J). Thus, L-WNT3A treatment improves theosteogenic capacity of autografts from aged animals.

Example 4

Expression of Wnt3a from Vector Specific for Serum-Free CHO Cells

Wnt3a secreting CHO suspension (CHO-S) cells could be also culturedunder serum-free conditions in suitable expression media, such asexpression media from Invitogen. A cGMP compatible CHO-S cells line, andtwo types of vectors to clone WNT3A were used. One vector was pOptivec,which is a TOPO® adapted bicistronic plasmid which allows rapid cloningof a gene containing a mammalian secretion signal and the gene ofinterest downstream of the CMV promoter. The dihydrofolate reductaseselection markers allows for rapid selection. This vector was used fortransient transfection of CHO-S cells. Another vector was pTargeT. Thisvector was used for transient transfection of CHO-S cells and also tocreate a stable cell line expressing WNT3A

Conditioned media (CM) from CHO cell cultures were collected between day3 and day 13 after induction and pooled. Based on analyses of proteinproduction in the CM on each day, this range of days was determined tobe optimal under the culture conditions. Under these conditions, highprotein production was observed to occur between days 3-13, while afterday 13 cells began to die.

Example 5

Replacement of Serum with a Lipid-Defined Substitute

The lipid components in serum are tested to determine whether they couldbe replaced with a defined set of lipids. Charcoal stripping removesnon-polar lipophilic materials (viruses, growth factors, hormones) fromserum. In comparison to control conditions where CHO cells are grown in10% FBS, CHO cells grown in charcoal-stripped FBS do not secrete Wnt3a.These data support the conclusion that a lipophilic component of serumis required for Wnt3a secretion. Supplementation with Lipid Mixture 1(Sigma) containing cholesterol, tocopherol, and non-animal derived fatty(inoleic, linolenic, myristic, oleic, palmitic and stearic) acids willimprove CHO cell growth rate and will restore Wnt3a secretion to CHOcells.

Example 6 Step-Wise Reduction in Serum Dependence

A sequential culturing as a means to reach a serum independent processfor WNT3A production is also tested. CHO cell growth rate is reducedsimultaneous with serum reduction; consequently, it is important toshift the time frame for the collection of conditioned media. In someinstances, cells are adapted to 0.5% FBS.

Although in the foregoing description the invention is illustrated withreference to certain embodiments, it is not so limited. Indeed, variousmodifications of the invention in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and fall within the scope of the appended claims.

What is claimed is:
 1. A method of preparing liposomal Wnt polypeptides,comprising: a) incubating Wnt polypeptides harvested from a conditionmedia comprising mammalian cells with a nonionic detergent selected fromNP-40, octyl glucoside, octyl thioglucoside, Triton X-100, Triton X-114,Tween 20, and Tween 80; b) purifying the Wnt polypeptides with a columnto generate purified Wnt polypeptides; and c) contacting the purifiedWnt polypeptides with an aqueous solution of liposomes, therebygenerating the liposomal Wnt polypeptides.
 2. The method of claim 1,wherein the nonionic detergent is octyl glucoside.
 3. The method ofclaim 1, wherein the nonionic detergent is octyl thioglucoside.
 4. Themethod of claim 1, wherein the liposome comprises1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC).
 5. The method ofclaim 1, wherein the liposome further comprises cholesterol.
 6. Themethod of claim 5, wherein the concentration of DMPC and cholesterol isdefined by a ratio of between about 70:30 and about 100:0 or betweenabout 70:30 and about 90:10.
 7. The method of claim 1, wherein thecondition media comprises Chinese hamster ovary (CHO) cells.
 8. Themethod of claim 1, wherein the condition media comprises up to 10% fetalbovine serum.
 9. The method of claim 1, wherein the purifying in step b)further comprises i) incubating the Wnt polypeptides on the column; andii) eluting the Wnt polypeptides with an elution buffer comprising asalt concentration of from about 150 mM to about 2M.
 10. The method ofclaim 1, wherein the column is immobilized with a sulfonatedpolyaromatic compound.
 11. The method of claim 1, wherein at least oneof the Wnt polypeptides comprises a lipid modification at an amino acidposition corresponding to amino acid residue 209 as set forth in SEQ IDNO:
 1. 12. The method of claim 11, wherein the lipid modification ispalmitoylation.
 13. The method of claim 12, wherein at least one of theWnt polypeptides is modified with palmitic acid.
 14. The method of claim1, wherein the Wnt polypeptides are mammalian Wnt polypeptides.
 15. Themethod of claim 1, wherein the Wnt polypeptides are Wnt3a polypeptides.16. The method of claim 15, wherein the Wnt3a polypeptides areintegrated into the liposomal membrane, protrude from the liposomalmembrane onto the outer surface of the lipid membrane, and are notincorporated into the aqueous core of the liposome.
 17. The method ofclaim 15, wherein the Wnt3a polypeptides comprise at least about 30%,about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, orabout 95% sequence identity to the amino acid sequence as set forth inSEQ ID NO: 1 or comprise the amino acid sequence at set forth in SEQ IDNO:
 1. 18. The method of claim 15, wherein the yield of the Wnt3apolypeptides after contacting with the aqueous solution of liposomes isbetween about 60% and about 90%.
 19. A liposomal Wnt polypeptidegenerated by the method of claim
 1. 20. The liposomal Wnt polypeptide ofclaim 19, wherein the liposomal Wnt polypeptide is liposomal Wnt3apolypeptide.
 21. A kit for generating bone graft materials comprisingthe liposomal Wnt polypeptide generated by the method of claim
 1. 22.The kit of claim 21, wherein the liposomal Wnt polypeptide is liposomalWnt3a polypeptide.